[ { "accession" : "PXD077396", "title" : "Mitochondrial surveillance activates the primary UPRmt", "projectDescription" : "The mitochondrial unfolded protein response (UPRmt) is critical for protecting mitochondria against proteotoxic stress. Current UPRmt models propose that mitochondrial defects are detected by cytosolic surveillance mechanisms after release of damage into the cytosol. However, these findings are based on models that induce rapid and severe mitochondrial dysfunction, and thus their mode of toxicity contrasts sharply from physiologically occurring mitochondrial damage, e.g. the gradual accumulation of reactive oxygen species (ROS) during age-related decline or chronic respiratory chain defects. Here, we employed a chemogenetic strategy that induces low levels of H2O2 in the mitochondrial matrix to investigate cellular responses to physiologically relevant levels of mitochondrial dysfunction. We uncover that this mild oxidative stress activates the UPRmt independently of cytosolic signals revealing a so far concealed primary surveillance mechanism within mitochondria. Moreover, we identify the mitochondrial presequence proteases MPP and Oct1 as early molecular targets of ROS: Oxidative stress induces glutathionylation of critical cysteine residues, resulting in diminished proteolytic activity and the accumulation of proteotoxic precursor aggregates in the matrix. These aggregates are detected by intramitochondrial surveillance systems, activating UPRmt signaling. Our findings uncover the primary response to mitochondrial dysfunction, and highlight the organelle's capacity for self-surveillance and its ability to initiate early and rapid protective signaling in the face of mitochondrial dysfunction.", "dataProcessingProtocol" : "MS/MS spectra were searched against the UniProt Saccharomyces cerevisiae reference proteome (UP000002311, downloaded February 2025), supplemented with custom FASTA sequences for Mas1, Mas2, and Oct1, as well as a curated contaminant database, using Proteome Discoverer 3.1 with the Sequest HT search engine. The precursor mass tolerance was set to 10 ppm, and the fragment ion mass tolerance was set to 0.02 Da. For spectra acquired using the EThcD method, b-, c-, y-, and z-type fragment ions were enabled during database searching. Trypsin was specified as the proteolytic enzyme. The following variable modifications were included: oxidation (M), glutathionylation (C), methionine loss (M), and combined methionine loss with N-terminal acetylation. Peptide quantification was performed using the precursor ion quantifier node in Proteome Discoverer, with summed precursor abundances used for protein-level quantification.", "sampleProcessingProtocol" : "Protein samples were digested using the Single Tube Solid Phase Sample Preparation (SP3) method on a KingFisher Apex platform, as described previously [cite: Voran, J.C.; Kilian, L.S.; Martini, S.; Luzarowski, M.; Noormalal, M.I.; Müller, O.J.; Rangrez, A.Y.; Frank, D. First Glance at Myeloid Leukaemia Factor 2 in Cardiomyocytes.]. To preserve potential cysteine S-glutathionylation, both reduction and alkylation steps were omitted. After digestion, peptides were desalted using Affinisep Stage Tips 200µl C18 T2. After initial sequential washing with (i) 50% MeOH, (ii) 80% ACN, 0.1% TFA, and (iii) 0.1% TFA, acidified peptides were loaded on the stage tips. After washing with 0.1% TFA, peptides were eluted twice using 80% ACN, 0.1% TFA. NanoLC–MS/MS analysis was performed using a Vanquish Neo system coupled to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). Peptides derived from recombinant Mas1, Mas2, and Oct1 were separated using a 30-min linear gradient from 4% to 32% solvent B over 25 min, followed by an increase to 49% solvent B over 5 min and a final wash at 99% solvent B. Each sample was analyzed in two independent LC–MS/MS runs. One run employed higher-energy collisional dissociation (HCD), and the second run employed electron-transfer/higher-energy collision dissociation (EThcD). For both acquisition strategies, the instrument was operated in data-dependent acquisition (DDA) mode. For HCD runs, MS1 spectra were acquired in the Orbitrap at a resolution of 120,000 (m/z 200), with an AGC target of 1.2 × 10⁶, maximum injection time of 50 ms, RF lens of 30%, and a scan range of m/z 400–1600. Dynamic exclusion was enabled for 10 s, excluding all charge states of a given precursor. Monoisotopic peak determination was enabled, and precursors were isolated using a 0.5 m/z window. Singly charged ions and ions with charge states above 5 were excluded, and the intensity threshold was set to 5 × 10³. MS2 spectra were acquired in the Orbitrap at a resolution of 30,000, with a maximum injection time of 54 ms, a custom AGC target of 1.25 × 10⁵, and a normalized collision energy (NCE) of 30%. For EThcD runs, MS1 spectra were acquired at a resolution of 120,000, with an AGC target of 1.2 × 10⁶, maximum injection time of 246 ms, RF lens of 36%, and a scan range of m/z 500–2000. Dynamic exclusion and monoisotopic peak determination were applied as described above. Singly charged ions and ions with charge states above 6 were excluded, and the intensity threshold was set to 5 × 10⁴. Precursors were isolated using a 1.6 m/z window. MS2 spectra were acquired in the Orbitrap at a resolution of 30,000 using EThcD fragmentation with a supplemental HCD activation energy of 25%, a maximum injection time of 54 ms, and a custom AGC target of 1.0 × 10⁵.", "projectTags" : [ ], "keywords" : [ "Presequence processing", "Reactive oxygen species", "Mitochondrial protein biogenesis", "Mitochondrial unfolded protein response" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-20", "updatedDate" : "2026-04-20", "submissionDate" : "2026-04-20", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Marcin Luzarowski" ], "labPIs" : [ "Marcin Luzarowski" ], "affiliations" : [ "Core Facility for Mass Spectrometry and Proteomics, Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany" ], "instruments" : [ "quadrupole ion trap orbitrap instrument" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Saccharomyces cerevisiae (Baker's yeast)" ], "organisms" : [ "Saccharomyces cerevisiae (baker's yeast)" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "EC-OT30-25-190-EThcD-11.raw", "EC-OT30-25-190-EThcD-05.raw", "EC-OT30-25-190-HCD-09.raw", "EC-OT30-25-190-EThcD-07.raw", "EC-OT30-25-190-EThcD-03.raw", "EC-OT30-25-190-HCD-13.raw", "EC-OT30-25-190-EThcD-01.raw", "EC-OT30-25-190-HCD-15.raw", "EC-OT30-25-190-HCD-05.raw", "EC-OT30-25-190-HCD-11.raw", "EC-OT30-25-190-EThcD-S01-S15_Trypsin_S_cerevisiae_cs.msf", "EC-OT30-25-190-EThcD-09.raw", "EC-OT30-25-190-HCD-07.raw", "EC-OT30-25-190-HCD-S01-S15_Trypsin_S_cerevisiae_cs.msf", "EC-OT30-25-190-HCD-01.raw", "EC-OT30-25-190-EThcD-15.raw", "EC-OT30-25-190-HCD-03.raw", "EC-OT30-25-190-EThcD-13.raw" ], "highlights" : { } }, { "accession" : "PXD077315", "title" : "Proteomic characterization of native and decellularized rat submandibular gland scaffolds prepared using the S3T1 protocol", "projectDescription" : "Salivary gland hypofunction lacks effective regenerative treatment, and organ-specific decellularized extracellular matrix scaffolds represent a promising biomaterial platform for tissue engineering. In this study, rat submandibular glands were decellularized using a rapid S3T1 workflow consisting of freeze-thaw pretreatment, 1% SDS treatment for 3 h, and 0.25% trypsin-EDTA treatment for 1 h. Native submandibular glands and decellularized scaffolds were compared by data-independent acquisition LC-MS/MS to characterize protein composition and extracellular matrix preservation. The dataset was generated to identify retained matrisome components and to evaluate the proteomic features of the decellularized scaffold in comparison with native tissue. These data support the use of the decellularized submandibular gland scaffold as a bioactive platform for salivary gland tissue engineering.", "dataProcessingProtocol" : "Proteomic analysis was performed using a data-independent acquisition workflow. Lyophilized native submandibular gland and decellularized scaffold samples were trypsin-digested and analyzed by LC-MS/MS. DIA-MS/MS data were processed using DIA-NN. Spectra were searched against the Rattus norvegicus reference proteome with a false discovery rate of 1%. Downstream analysis was performed to characterize protein composition, identify retained extracellular matrix proteins, and classify matrisome components based on MatrisomeDB annotation. Functional enrichment analyses, including Gene Ontology and KEGG pathway analysis, were performed using DAVID Bioinformatics Resources.", "sampleProcessingProtocol" : "Rat submandibular glands were harvested immediately after euthanasia and briefly rinsed in cold PBS. Tissues were subjected to three freeze-thaw cycles by rapid freezing in liquid nitrogen followed by thawing at 37°C. For decellularization, each gland was immersed in 1% SDS for 3 h on an orbital shaker at 200 rpm, followed by three sequential PBS washes of 10 min each. Samples were then treated with 0.25% trypsin-EDTA at 37°C for 1 h and washed again three times in PBS under the same conditions. The resulting decellularized scaffolds were sterilized, washed thoroughly, lyophilized, and prepared for downstream proteomic analysis. Native rat submandibular glands were processed in parallel as controls. For proteomics, samples from native SMG and decellularized SMG scaffold groups were collected with three biological replicates per group.", "projectTags" : [ ], "keywords" : [ "Extracellular matrix scaffold", "Rat", "Proteomics", "Decellularization", "Submandibular gland", "Lc-ms/ms", "Salivary gland tissue engineering", "Dia" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-21", "updatedDate" : "2026-04-17", "submissionDate" : "2026-04-17", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Jie Gao" ], "labPIs" : [ "Jie Gao" ], "affiliations" : [ "Shanghai Jiao Tong University" ], "instruments" : [ "quadrupole orbitrap astral instrument" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Rattus norvegicus (Rat)" ], "organisms" : [ "Rattus norvegicus (rat)" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-independent acquisition" ], "sdrf" : "", "projectFileNames" : [ "FWMS20250734_S3T1-3.raw", "FWMS20250734_Native2.raw", "report.pr_matrix.tsv", "checksum.txt", "report.pg_matrix.tsv", "FWMS20250734_Native3.raw", "FWMS20250734_S3T1-1.raw", "FWMS20250734_S3T1-2.raw", "FWMS20250734_Native1.raw" ], "highlights" : { } }, { "accession" : "PXD077306", "title" : "GPATCH11 ortholog Sap34 regulates pre-mRNA splicing by interacting with early spliceosomal complexes in Schizosaccharomyces pombe", "projectDescription" : "Pre-mRNA splicing is an essential step in gene expression regulation. It is mediated by the spliceosome, a large ribonucleoprotein complex that undergoes dynamic structural and compositional rearrangements during each splicing cycle. Although the mechanisms of splicing and the roles of main spliceosomal components are well defined, the identities and functions of transiently associated spliceosomal proteins remain incompletely understood. Here, we investigated the molecular function of the poorly characterized G-patch domain–containing protein SPAC6F6.19 (herein Sap34, for spliceosome-associated protein of 34 kDa) in Schizosaccharomyces pombe, an ortholog of human GPATCH11. Using affinity purification and a yeast two-hybrid assay, we analyzed its interactome and identified its interaction partners. In addition, long-read sequencing was employed to assess Sap34-dependent changes in splicing efficiency. We found that Sap34 forms a complex with components of the U2 small nuclear ribonucleoprotein (snRNP) and the U4/U6 × U5 tri-snRNP, which are required for early spliceosome assembly and activation. Furthermore, we defined the interaction specificity of Sap34 with other splicing proteins, demonstrating the importance of its C-terminal region for binding to Sap61 and Ini1, and of its G-patch domain for interaction with the ATP-dependent RNA helicase Prp43. Notably, we showed that deletion of sap34 leads to a global reduction in splicing efficiency, predominantly associated with increased intron retention. Together, these findings identify Sap34 as a previously unrecognized and important G-patch domain–containing protein regulating the early steps of pre-mRNA splicing in fission yeast.", "dataProcessingProtocol" : "The resulting datasets were processed together using MaxQuant version 1.6.17.0 with the built-in Andromeda search engine [49]. The searched database consisted of S. pombe proteomes (UniProt, downloaded 24.1.2025, isoforms included and PomBase, downloaded 23.1.2025) and a predicted N-terminally extended Sap34 amino acid sequence (with TAP tag) based on the newly defined sequence of the sap34 allele. The cleavage specificities of proteases were set as follows: trypsin – C-terminal of R and K also if P follows; GluC – C-terminal of E and D; chymotrypsin – C-terminal of F, Y, L, W, and M. The number of tolerated missed cleavages was 2 for trypsin, 4 for GluC, and 3 for chymotrypsin. Carbamidomethylation (C) was set as a permanent modification, and oxidation (M), acetylation (protein N-terminus), and phosphorylation (STY) were set as variable modifications. The precursor ion mass tolerance was set to 20 ppm in the first search and to 4.5 ppm in the main search upon recalibration, and the fragment tolerance was 20 ppm. The target-decoy database strategy was used to control for false identifications, and the false discovery rate of 1% was imposed at both the peptide-spectrum-match and protein group levels.", "sampleProcessingProtocol" : "The samples were reduced with 5 mM DTT, then alkylated with 15 mM iodoacetamide, and the reaction was quenched with an additional 5 mM DTT. Parallel digestions with trypsin, GluC, and chymotrypsin were performed overnight (16 h) in the presence of phosphatase inhibitors at 37 °C, 37 °C, and 25 °C, respectively. Digestions were further processed and measured separately. Peptides were purified by microtip C18 SPE. The separation of peptides was performed by nano-HPLC Dionex UltiMate 3000 RSLCnano system (Thermo Fisher Scientific). The samples were loaded onto a trap column (PepMap100 C18, 300 μm × 5 mm, 5-μm particle size; Thermo Fisher Scientific) and separated by an EASY-Spray C18 analytical column with integrated nanospray emitter (PepMap RLSC C18, 75 μm × 500 mm, 2-μm particle size, Thermo Fisher Scientific) using a 60-min gradient (3–43% B) at a flow rate of 250 nL/min. The two mobile phases used were: 0.1% formic acid (v/v) and 80% acetonitrile (v/v) with 0.1% formic acid. Eluted peptides were sprayed directly into the Orbitrap Elite mass spectrometer (ThermoFisher Scientific). The emitter potential was set to 2.3 kV, the transfer capillary temperature to 250 °C, and the S-lens RF level to 60%. Spectral datasets were collected in a data-dependent mode using the Top15 strategy to select precursor ions for fragmentation [48]. Precursors were measured in the mass range 300–1,700 m/z with a resolution of 120,000, and fragmented by the HCD mechanism with a normalized collision energy of 25. MS/MS scans were acquired at a resolution of 15,000. Each sample was measured in technical duplicates.", "projectTags" : [ ], "keywords" : [ "G-patch protein; sap34; spac6f6.19; splicing factor; pre-mrna splicing; fission yeast" ], "doi" : "10.6019/PXD077306", "submissionType" : "COMPLETE", "publicationDate" : "2026-04-21", "updatedDate" : "2026-04-17", "submissionDate" : "2026-04-17", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Peter Barath" ], "labPIs" : [ "Peter Barath" ], "affiliations" : [ "Institute of Chemistry, SAS" ], "instruments" : [ "LTQ Orbitrap Elite" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Schizosaccharomyces pombe 927" ], "organisms" : [ "Schizosaccharomyces pombe 927" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "SPAC6F6-19_P1_T_01.raw", "mzTab.mzTab", "SPAC6F6-19_P1_T_02.raw", "SPAC6F6-19_P3_G_01.raw", "SPAC6F6-19_P3_CH_01.raw", "SPAC6F6-19_P2_T_02.raw", "SPAC6F6-19_P3_CH_02.raw", "APL_files.zip", "SPAC6F6-19_P3_G_02.raw", "SPAC6F6-19_P2_T_01.raw" ], "highlights" : { } }, { "accession" : "PXD077187", "title" : "Establishment and application of a vesicle extraction method for clinical strains of Pseudomonas aeruginosa", "projectDescription" : "Pseudomonas aeruginosa is a versatile pathogen capable of causing illnesses that range from mild infections to life-threatening conditions. Its virulence is driven by a wide array of factors, among which extracellular vesicles (EVs) have gained recognition as important contributors to its pathogenicity. Despite this, the full scope of their roles remains unclear. A major barrier to EV characterization is the difficulty of vesicle isolation—procedures are often lengthy, yield is low, and specialized equipment is required. In this study, we assessed the effectiveness of a rapid vesicle extraction method from clinical strains of P. aeruginosa. To that end, we first selected and characterized six phenotypically diverse clinical strains of P. aeruginosa (two reference strains and 4 clinical isolates, including one strain from a cystic fibrosis patient) and used them to evaluate the vesicle extraction method. The results obtained through SDS-PAGE analysis, western blot, protein quantification, TEM, dynamic light scattering and mass spectrometry indicated the presence of vesicles in all samples; however, it was also possible to observe a large number of contaminants in some of them (mainly LS07 and Z37). Subsequent treatment with enzymes (DNase and/or alginate lyase) allowed for the elimination of the contaminants as observed by electron microscopy. Our results suggest that the method is suited for the vesicle extraction of clinical isolates of P. aeruginosa, where traditional methods, such as ultracentrifugation, are not available The phenotypic complexity of these strains presents challenges that current rapid purification methods are ill-equipped to handle, highlighting the need for improved or alternative approaches.", "dataProcessingProtocol" : "Raw mass spectrometry data were analyzed using MaxQuant (version 2.4.14.0; Cox & Mann, 2008). Spectra were searched against the Pseudomonas reference proteome (UniProt, UP000002438), Default search parameters were used.", "sampleProcessingProtocol" : "Proteins present in vesicles samples were reduced with 2 mM dithiothreitol (DTT) for 30 minutes at 37 °C, followed by alkylation with 5 mM iodoacetamide (IAA) for 30 minutes at room temperature in the dark. Proteins were digested overnight at 37 °C with trypsin at a 1:50 (w/w) enzyme-to-substrate ratio. Resulting peptides were desalted using home-made C18 StageTips, as described by Rappsilber et al. (2003). Peptides were analyzed on a nanoElute2 LC system (Bruker Daltonics) coupled to a timsTOF HT mass spectrometer (Bruker Daltonics) equipped with a CaptiveSpray nano-electrospray ionization (nano-ESI) source. Chromatographic separation was performed using a PepMap Neo trap column (C18, 300 µm × 5 mm, Thermo Fisher Scientific) and a PepSep Ultra analytical column (C18, 25 cm × 75 µm, 1.5 µm particle size, Bruker Daltonics). Peptides were eluted with a linear gradient from 5% to 37% acetonitrile (v/v) over 30 minutes at a flow rate of 250 nL/min, with the column maintained at 50 °C. Data-dependent acquisition (DDA) was carried out using the “DDA PASEF-standard_1.1.sec_cycletime.m” method with default instrument parameters.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-16", "updatedDate" : "2026-04-15", "submissionDate" : "2026-04-15", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Serena Schwenkert" ], "labPIs" : [ "Serena Schwenkert" ], "affiliations" : [ "MSBioLMU Großhaderner Str. 2-4 82152 Planegg-Martinsried" ], "instruments" : [ "timsTOF HT" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Pseudomonas aeruginosa" ], "organisms" : [ "Pseudomonas aeruginosa" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "LS07_3_Slot1-24_1_3962.d.zip", "LS06_2_Slot1-20_1_3957.d.zip", "PAO1_2_Slot1-11_1_3945.d.zip", "PAO1_ultracent_2_Slot1-29_1_3969.d.zip", "LS06_1_Slot1-19_1_3956.d.zip", "ATCC27853_2_Slot1-14_1_3949.d.zip", "LS07_1_Slot1-22_1_3960.d.zip", "LS03_1_Slot1-16_1_3952.d.zip", "LS06_3_Slot1-21_1_3958.d.zip", "ATCC27853_1_Slot1-13_1_3948.d.zip", "checksum.txt", "PAO1_3_Slot1-12_1_3946.d.zip", "Z37_1_Slot1-25_1_3964.d.zip", "combined.zip", "Z37_3_Slot1-27_1_3966.d.zip", "LS07_2_Slot1-23_1_3961.d.zip", "LS03_2_Slot1-17_1_3953.d.zip", "PAO1_1_Slot1-10_1_3944.d.zip", "LS03_3_Slot1-18_1_3954.d.zip", "PAO1_ultracent_3_Slot1-30_1_3970.d.zip", "ATCC27853_3_Slot1-15_1_3950.d.zip", "Z37_2_Slot1-26_1_3965.d.zip", "PAO1_ultracent_1_Slot1-28_1_3968.d.zip" ], "highlights" : { } }, { "accession" : "PXD077093", "title" : "Proximity interactions of Frizzled4 (FZD4) in the bEnd.3 mouse brain endothelial cell line", "projectDescription" : "Frizzled4 (FZD4) is a pivotal molecule in the CNS vasculature and a target for therapeutic intervention. Little is known how FZD4 is regulated. A proximity biotinylation screen with FZD4 as bait in a mouse brain endothelial cell line was performed. The goal is to identify candidate regulators of FZD4.", "dataProcessingProtocol" : "We processed peptide tandem MS using SEQUEST (Thermo Scientific) in Proteome Discoverer 4.0. The mouse Universal Proteome database (UP000000589) was downloaded from UniProt on 11/19/2021 and merged with a common lab contaminant protein database (https://www.thegpm.org/crap/) (55,214 total protein sequences). We applied the precursor mass recalibration node with precursor mass tolerance 20 ppm, product ion tolerance 0.2 Da with fixed carbamidomethyl (CAM) modification of cysteine 57.0215 m/z. The SEQUEST (PMID: 24226387) database search parameters included enzyme trypsin full specificity, 2 missed cleave sites; precursor tolerance 15 ppm, fragment ion tolerance 0.05 Da and maximum 4 dynamic modifications per peptide. We specified CAM cysteine (+57.021 Da) as a fixed modification and the dynamic modifications were acetylation of protein N-terminus (+42.011 Da), oxidation of methionine (+15.995 Da), conversion of glutamine to pyroglutamic acid (-17.027 Da), methionine loss at the protein N-terminus (-131.040 Da), methionine loss + acetylation at the protein N-terminus (-89.030 Da), biotinylation of lysine (226.078 Da) and deamidation of asparagine or glutamine (+0.984 Da). We applied 1% protein and peptide False Discovery Rate (FDR) filters using the Percolator algorithm (https://doi.org/10.1038/nmeth1113) in PD. We used the label free quantification workflow in PD 2.4 that included steps for feature extraction, chromatographic alignment, peptide mapping to features, protein abundance calculation, normalization, protein relative abundance ratio calculation and hypothesis testing for significance of relative fold chance. We applied the Minora Feature Detector algorithm in PD 2.3 for the assignment of chromatographic features for isotopically related peaks within a 0.2 minute retention time range. After features were assigned, chromatographic alignment was performed; the file with the largest number of features was assigned as the reference to which features from each file were aligned within a 4 minute (RT) window tolerance and 12 ppm mass tolerance. Retention times were adjusted from a non-linear regression-based fit of the differences in retention time between reference and sample. Peptide were mapped to retention time-aligned consensus features across samples with the requirement that at least one sample contains a peptide spectral match. We applied total peptide summed normalization across samples. Protein abundance calculations were made from summed peptide abundances. We used the background t-test for hypothesis testing with the null hypothesis of equal protein abundance. Protein relative abundances between groups were reported with p-values adjusted with the Benjamini-Hochberg method for multiple testing corrections.", "sampleProcessingProtocol" : "bEnd.3 cells were virally transduced with vectors encoding V5-FZD4-BioID or GFP-BioID as well as blasticidin resistance. After selection, the stable populations were used for a proximity biotinylation screen, for which GFP-BioID cells served as negative control. Four 150 cm2 dishes per sample were incubated for 24 hours with 50 µM biotin. Duplicate samples were generated. Cells were washed in PBS and directly lysed. Cells were lysed in 8 M urea 50 mM Tris, pH 7.4, buffer with protease inhibitor (87785; Thermo Fisher Scientific) and DTT under sonication, precleared with gelatin-sepharose beads (17095601; Cytiva), incubated with streptavidin-sepharose beads (17511301; Cytiva/GE Healthcare), washed in 8 M urea, 50 mM Tris, pH 7.4, and resuspended in 50 mM ammonium bicarbonate saturated with 1 mM biotin. Samples were reduce-alkylated in 8 M urea, 50 mM ammonium bicarbonate buffer with 10 mM tris(2-carboxyethyl)phosphine (TCEP) at 30 °C for 60 min and 30 mM iodoacetamide (IAA) in the dark at room temperature for 30 min. Samples were diluted to 1M urea and digested with mass spec grade Trypsin/LysC mix overnight (V5071; Promega). The peptide eluates were acidified with formic acid and purified via a C18 matrix (5190-6532; Agilent) using Agilent AssayMap BRAVO liquid handling system. Solvent was removed in a SpeedVac and subjected to LC-MS/MS. We reconstituted the dried peptide mixtures in 98:2:0.1,H2O:acetonitrile (ACN):formic acid (FA) and analyzed ~200 nanogram of each sample by capillary LC-MS on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA) online with a Thermo UltiMate™ 3000 RSLCnano LC system. Peptides were separated on a 40 cm self-packed C18 capillary column with 100 um inner diameter, with Dr. Maisch GmbH ReproSil-PUR 12 Å C18-AQ, 1.9 um particle size; the column was maintained at 55°C with a column heater from Sonation (Biberach, Germany). Peptides were loaded directly on column at 325 nl/min with 98:2:0.1,H2O:ACN:FA. We performed elution of the peptides with the following gradient: 5 – 32% solvent B from 0 – 50 minutes, 32 – 45% solvent B from 50 – 55 minutes, 45 – 90% solvent B from 55 – 60 minutes 325 nl/minute, where solvent A was 0.1% formic acid in water and solvent B was 0.1% formic acid in ACN. We operated the mass spectrometer with the following parameters: ESI voltage 2.1kV, ion transfer tube 275 °C; Orbitrap MS1 scan 120k resolution in profile from 380 – 1580 m/z with 50 msec injection time and 120% normalized automatic gain control (AGC); MS2 triggered on precursors charge states 2 – 5 above 20000 counts; precursor isolation window 1.6 Da; MIPS (monoisotopic peak determination) set to Peptide; higher energy collisional dissociation (HCD) at 35% with MS2 Orbitrap detection at 50k resolution (at 200 m/z), 86 msec injection time, 100% (1000) AGC, dynamic exclusion duration 15 sec with +/- 10 ppm mass tolerance.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "10.6019/PXD077093", "submissionType" : "COMPLETE", "publicationDate" : "2026-04-14", "updatedDate" : "2026-04-13", "submissionDate" : "2026-04-13", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Kevin Murray" ], "labPIs" : [ "Harald Junge" ], "affiliations" : [ "Associate Professor and Research Director Department of Ophthalmology and Visual Neuroscience University of Minnesota" ], "instruments" : [ "Orbitrap Fusion" ], "softwares" : [ ], "quantificationMethods" : [ "Relative quantification" ], "sampleAttributes" : [ "cell culture", "endothelial cell", "Mus musculus (Mouse)" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Cell culture", "Endothelial cell" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "junge_jo000024_20210119_17951_BioID_FZD4_1.raw", "junge_jo000024_20210119_17951_BioID_GFP1.raw", "junge_jo000024_20210119_17951_BioID_FZD4_2.raw", "junge_jo000024_20210119_17951_BioID_GFP2_pt2.raw", "junge_jo000024_20210119_17951_BioID_LFQ_ANOVA.mzTab" ], "highlights" : { } }, { "accession" : "PXD077080", "title" : "Adaptation of an immunoaffinity chromatography protocol for the isolation of the cell line immunopeptides", "projectDescription" : "BACKGROUND. For developing targeted immunotherapy against ovarian adenocarcinoma, direct identification of peptides presented by major histocompatibility complex class I (HLA I) on the surface of tumor cells can be utilized. However, standard immunopeptidomics protocols may vary substantially in their efficiency of immunopeptidome isolation depending on lysis conditions. Optimizing detergents for immunoaffinity purification of complexes enhances analytical sensitivity, expands the repertoire of identified ligands, and increases the likelihood of detecting clinically relevant peptides suitable for personalized immunotherapeutic approaches.\nAIM. To evaluate the effect of various detergents on the efficiency of identification of peptide ligands of HLA I complexes. \nMETHODS. Immunopeptidome from cell lines were isolated using immunoaffinity chromatography and analyzed by liquid chromatography-mass spectrometry.\nRESULTS. In this study, we tested an affinity chromatography-based immunopeptidome isolation protocol, comparing various detergents (CHAPS, NP-40, SOD, and Triton X-100) for cell lysis, and observed no statistically significant differences in the number of identified peptides.\nCONCLUSION. Each of the four tested detergents provides identification of unique sets of peptides.", "dataProcessingProtocol" : "Raw LC-MS/MS data from the Q Exactive HF-X mass spectrometer were converted to .mgf peak lists with MSConvert (ProteoWizard Software Foundation). For this procedure, we used the following parameters: “--mgf --filter peakPicking true [1,2]”. For a thorough protein identification, the generated peak lists were searched with MASCOT (version 2.5.1, Matrix Science Ltd., UK) and X! Tandem (ALANINE, 2017.02.01, The Global Proteome Machine Organization) against the UniProt Knowledgebase (taxon human) with the concatenated reverse decoy dataset. The precursor and fragment mass tolerance were set at 20 ppm and 50 ppm, respectively. The database search parameters included no enzyme or possible post-translational modification (PTM). For X! Tandem, we also selected parameters that allowed us to quickly check for protein N-terminal acetylation, peptide N-terminal glutamine ammonia loss or peptide N-terminal glutamic acid water loss.", "sampleProcessingProtocol" : "Cell lines used.\nThe Jurkat (ATCC, TIB-152), K-562 (ATCC, CCL-243, USA) and HB-95 hybridoma cell lines (ATCC, HB-95) were used in the study. Jurkat and K-562 cell lines were cultured in RPMI 1640 medium (PanEco, C330p, Russia) 10% FBS (Gibco, 26140079, USA), 50 units/ml gentamicin (Dalhimfarm, Russia), 1.6 mM glutamine (GlutaMAX, Gibco, 35050061). All cell lines were cultured to the required number, then centrifuged for 5 min at 150 g (Eppendorf 5804 R, USA). The cell pellet was washed three times with Dulbecco's phosphate buffered saline, pH 7.4 (PanEco, P060). The supernatant was collected, and the cell pellet was stored at -80°C until further experiments. To produce antibodies to HLA I, HB-95 hybridoma cells (producer of HLA-ABC w6/32 antibodies, ATCC, HB-95) were cultured. Primary culture was performed in DMEM F12 Advanced medium (Gibco, 12634010) supplemented with 10% FBS (inactivation at 56°C for 30 minutes), 50 units/ml gentamicin, and 1.6 mM glutamine. When the cell density reached 600,000 cells/mL, the medium was replaced with one containing half as much FBS. This gradually reduced the FBS concentration in the medium. Once the FBS concentration reached 0.5%, the medium was replaced with HyClone ADCF-mab (Cytiva, SH30349.02, USA), 50 U/mL gentamicin, and 1.6 mM glutamine. Cells were cultured in this medium until 80-90% of the cells were killed, after which the supernatant containing antibodies was collected. Antibody concentrations in the cell medium were measured using the Easy-Titer mouse IgG Assay (Thermo Scientific, 23300, USA) according to the manufacturer's recommendations.\nAll cells were regularly tested for mycoplasma contamination and cultured at 37°C and 5% CO2.\n\nPreparation of cell line lysates.\nWhen working with cell lines, 5 ml of lysis buffer (150 mM NaCl, 50 mM Tris-HCl, Protease Inhibitor Cocktail X100 (Thermo Scientific, 78438), and detergent) were added to the dried cell pellet and incubated for 1 hour on a multirotator (BioSan Multi-Bio RS-24) at 4°C. For lysis of the K-562 cell line, 1% NP-40 was used. Detergent concentrations were selected based on literature data, but were not less than twice the critical micelle concentration (CMC). The cell lysate was sonicated for 5 min (Elma S30H), then centrifuged for 15 min at 14,000 g and 4°C (Eppendorf 5804 R, rotor A-4-44), and the supernatant was collected.\n\npHLA I Immunoprecipitation.\nFor isolation, 1 ml of Pierce Protein A/G Agarose resin suspension (Thermo Fisher, 20422) was placed on a low-pressure chromatography column (Econo-Column 0.5x5 cm, Bio-Rad, USA). The resin was washed with 50 ml of phosphate-buffered saline (PBS, pH 7.4) at a flow rate of 1 ml/min. Cell medium obtained after culturing the HB-95 hybridoma was then loaded onto the column at a concentration of 2 mg of antibody per ml of resin. The cell medium was diluted 1:1 with PBS before loading. The column was washed with 5 ml of 150 mM NaCl and 50 mM Tris-HCl, after which the cell lysate was added and incubated for 2.5 hours using a peristaltic pump (Longerpump BT100-1L, Russia). The column was then washed sequentially with 50 ml of PBS, 5 ml of 150 mM NaCl, 50 mM Tris-HCl, and 10 ml of mQ, and eluted with 10 ml. pHLA I and antibody complexes were eluted with 10% acetic acid. After completion of the column run, it was washed with 50 ml of PBS and then preserved in PBS with 0.02% NaN3.\nThe eluate was concentrated using a Concentrator plus centrifugal vacuum evaporator (Eppendorf, USA) at 60°C for 4.5 hours to 100–200 µl. The concentrated eluate was diluted with a solution of 3% acetonitrile in 0.1% TFA to 500 µl and sonicated for 5 minutes (Elma S30H ultrasonic bath). The peptides were then separated from the HLA complexes using centrifugal filtration cartridges (Vivaspin 500, 10 kDa) according to the manufacturer's protocol. The cartridges were additionally washed twice with 300 µl of 3% acetonitrile in 0.1% TFA. The peptides were dried in a Concentrator plus vacuum evaporator (Eppendorf) at 60°C for 6 hours and stored at -80°C until chromatograph mass spectrometry analysis.\n\nChromatographic mass spectrometry analysis of HLA I ligands.\nMass spectrometric analysis was performed on a Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). The chromatographic system was assembled using a pre-column-column scheme (column: sorbent - Aeris Peptide XB-C18 (Agilent, USA), length - 15 cm, internal diameter - 75 μm, particle size - 3 μm, pore diameter - 120 A, pre-column: sorbent - Aeris Peptide XB-C18, (Agilent, USA), length - 1 cm, internal diameter - 100 μm, particle size - 3 μm, pore diameter - 120 A, solvent A - 98.9% H2O, 1% MeOH, 0.1% formic acid; solvent B - 99.9% ACN, 0.1% formic acid), the chromatographic columns were thermostatted at 50°C. On the pre-column, application and washing were carried out for 10 minutes at a flow rate of 2 μl/min of solvent A. Separation was carried out at 300 nl/min, gradient: 1 min – 95% solvent A, 5% solvent B; The acetonitrile content was then increased linearly over 120 minutes to 60% solvent A and 40% solvent B. The chromatographic system was then washed with 95% solvent B for 10 minutes, followed by a 17-minute wash with 95% solvent A and 5% solvent B. Mass spectral acquisition mode: mass spectral acquisition time – 250 ms, mass/charge ratio range from 300 to 1250 m/z, criteria for preferential selection of ions for isolation and fragmentation – charge 2-5, the number of parent ions sampled in each cycle – no more than 50, minimum intensity – 100 counts/s. After the first analysis, the ion was forcibly excluded from the list of ions for fragmentation for 15 s. Fragmentation parameters: fragmentation spectra accumulation time – 100 ms, mass/charge ratio range from 100 to 2000 Th, voltage in the collision cell varied linearly from 25 V to 55 V, full cycle time – no more than 5.3 s.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "10.6019/PXD077080", "submissionType" : "COMPLETE", "publicationDate" : "2026-04-15", "updatedDate" : "2026-04-13", "submissionDate" : "2026-04-13", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Georgij Arapidi" ], "labPIs" : [ "Georgij Arapidi" ], "affiliations" : [ "Head of System Biology Lab. at Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency" ], "instruments" : [ "Q Exactive HF" ], "softwares" : [ ], "quantificationMethods" : [ "Spectrum counting" ], "sampleAttributes" : [ "cell culture", "Homo sapiens (Human)" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Cell culture" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Immunopeptidomics", "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "P1035.mgf", "P1078.mgf", "P1043.mgf", "P1036.dat", "P1078.tandem.xml", "P1049.mzid", "P1035.mzid", "P1035.tandem.xml", "P1035.mzid_P1035.MGF", "P1039.mzid_P1039.MGF", "checksum.txt", "P1042.mzid_P1042.MGF", "P1046.mzid_P1046.MGF", "P1034.dat", "P1048.tandem.xml", "P1043.raw", "P1050.dat", "P1078.mzid", "P1039.raw", "P1077.dat", "P1046.dat", "P1032.tandem.xml", "P1032.dat", "P1075.dat", "P1048.mzid", "P1034.mzid", "P1076.mgf", "P1042.tandem.xml", "P1033.mgf", "P1050.mzid", "P1074.dat", "P1049.dat", "P1041.tandem.xml", "P1041.raw", "P1040.dat", "P1050.raw", "P1032.mzid", "P1076.raw", "P1033.raw", "P1039.mgf", "P1047.dat", "P1047.mzid", "P1048.mgf", "P1048.raw", "P1041.mgf", "P1078.raw", "P1041.mzid", "P1042.dat", "P1076.mzid", "P1035.raw", "P1043.mzid", "P1036.mgf", "P1034.mgf", "P1033.mzid_P1033.MGF", "P1040.tandem.xml", "P1078.dat", "P1035.dat", "P1040.mzid_P1040.MGF", "P1048.mzid_P1048.MGF", "P1074.mzid", "P1046.raw", "P1043.dat", "P1050.tandem.xml", "P1075.mzid", "P1033.tandem.xml", "P1076.dat", "P1075.mgf", "P1032.mgf", "P1033.dat", "P1039.tandem.xml", "P1049.tandem.xml", "P1043.tandem.xml", "P1077.mgf", "P1074.raw", "P1050.mgf", "P1046.mgf", "P1042.mzid", "P1074.tandem.xml", "P1075.raw", "P1040.mzid", "P1032.raw", "P1049.mgf", "P1046.mzid", "P1047.mgf", "P1047.tandem.xml", "P1036.mzid_P1036.MGF", "P1034.mzid_P1034.MGF", "P1032.mzid_P1032.MGF", "P1048.dat", "P1039.dat", "P1043.mzid_P1043.MGF", "P1047.mzid_P1047.MGF", "P1077.mzid", "P1077.raw", "P1041.mzid_P1041.MGF", "P1040.mgf", "P1049.mzid_P1049.MGF", "P1050.mzid_P1050.MGF", "P1034.tandem.xml", "P1077.tandem.xml", "P1042.raw", "P1074.mgf", "P1036.mzid", "P1033.mzid", "P1039.mzid", "P1076.tandem.xml", "P1075.mzid_P1075.MGF", "P1078.mzid_P1078.MGF", "P1074.mzid_P1074.MGF", "P1076.mzid_P1076.MGF", "P1077.mzid_P1077.MGF", "P1034.raw", "P1047.raw", "P1042.mgf", "P1036.raw", "P1075.tandem.xml", "P1049.raw", "P1041.dat", "P1036.tandem.xml", "P1040.raw", "P1046.tandem.xml" ], "highlights" : { } }, { "accession" : "PXD077095", "title" : "Identification of major histocompatibility complex class I ligands in ovarian adenocarcinoma tumors", "projectDescription" : "BACKGROUND. For developing targeted immunotherapy against ovarian adenocarcinoma, direct identification of peptides presented by major histocompatibility complex class I (HLA I) on the surface of tumor cells can be utilized.\nAIM. To identify ligands of HLA I of ovarian adenocarcinoma tumors. \nMETHODS. Immunopeptidome from postoperative material of patients with ovarian adenocarcinoma were isolated using immunoaffinity chromatography and analyzed by liquid chromatography-mass spectrometry.\nRESULTS. Using NP-40, 5 peptides belonging to the proteins of cancer/testis antigens (CTA) were identified in the immunopeptidomes of postoperative material from patients with ovarian adenocarcinoma, 3 of which, according to the human protein atlas, are indeed not expressed in normal ovarian tissues.\nCONCLUSION. Immunopeptidome analysis allows the identification of peptides of CTA proteins, but a larger sample of postoperative material from patients with ovarian adenocarcinoma and experimental testing of the immunogenicity of the identified peptides are needed for further research.", "dataProcessingProtocol" : "Raw LC-MS/MS data from the Q Exactive HF-X mass spectrometer were converted to .mgf peak lists with MSConvert (ProteoWizard Software Foundation). For this procedure, we used the following parameters: “--mgf --filter peakPicking true [1,2]”. For a thorough protein identification, the generated peak lists were searched with MASCOT (version 2.5.1, Matrix Science Ltd., UK) and X! Tandem (ALANINE, 2017.02.01, The Global Proteome Machine Organization) against the UniProt Knowledgebase (taxon human) with the concatenated reverse decoy dataset. The precursor and fragment mass tolerance were set at 20 ppm and 50 ppm, respectively. The database search parameters included no enzyme or possible post-translational modification (PTM). For X! Tandem, we also selected parameters that allowed us to quickly check for protein N-terminal acetylation, peptide N-terminal glutamine ammonia loss or peptide N-terminal glutamic acid water loss.", "sampleProcessingProtocol" : "Tumors used.\nThis study utilized ovarian adenocarcinoma tumor tissue samples from patients who had undergone chemotherapy with taxanes and carboplatin (n = 3, patients 1-3) and without prior treatment (n = 1, patient 4). Samples were obtained from the Russian Research Center of Roentgenology and Radiology (Moscow, Russia). Diagnoses were confirmed by morphological examinations. The study was approved by the Ethics Committee of the Russian Research Center of Roentgenology and Radiology (Agreement and Protocol No. 30-2018/E dated November 13, 2018). All patients provided written consent to participate.\n\nPreparation of tissue lysates.\nFor tumor tissue, a 0.2 g sample was homogenized with ceramic beads (MagNA Lyser Green Beads, Roche, 03358941001, Switzerland) in 1 ml of lysis buffer (150 mM NaCl, 50 mM Tris-HCl, Protease Inhibitor Cocktail X100, 1% NP-40) using an automated homogenizer (MagNA Lyser, Roche) at 7000 g with periodic sample cooling. The tissue homogenate, along with the ceramic beads, was transferred to 4 ml of lysis buffer and left in a multirotator (BioSan Multi-Bio RS-24) for 1 hour at 4°C. The resulting tissue lysate was pre-sonicated for 5 minutes (Elma S30H ultrasonic bath), centrifuged for 15 minutes at 14,000 g and 4°C (Eppendorf 5804 R, rotor A-4-44), and the supernatant was collected.\n\npHLA I immunoprecipitation.\nFor isolation, 1 ml of a Pierce Protein A/G Agarose resin suspension (Thermo Fisher, 20422) was loaded onto a low-pressure chromatography column (Econo-Column 0.5x5 cm, Bio-Rad, USA). The resin was washed with 50 ml of phosphate-buffered saline (PBS, pH 7.4) at a flow rate of 1 ml/min. Cell medium obtained after culturing the HB-95 hybridoma was then loaded onto the column at a concentration of 2 mg of antibody per ml of resin. Before loading, the cell medium was diluted 1:1 with PBS. The column was washed with 5 ml of 150 mM NaCl and 50 mM Tris-HCl. The tumor lysate was then added and incubated for 2.5 hours using a peristaltic pump (Longerpump BT100-1L, Russia). The column was then washed sequentially with 50 ml of PBS, 5 ml of 150 mM NaCl, 50 mM Tris-HCl, and 10 ml of mQ, and eluted with 10 ml. pHLA I and antibody complexes were eluted with 10% acetic acid. After completion of the work, the column was washed with 50 ml of PBS and then preserved in PBS with 0.02% NaN3. The eluate was concentrated using a Concentrator plus centrifugal vacuum evaporator (Eppendorf, USA) at 60°C for 4.5 hours to 100–200 μl. The concentrated eluate was diluted with a solution of 3% acetonitrile in 0.1% TFA to 500 μl and sonicated for 5 min (Elma S30H ultrasonic bath). The peptides were then separated from the HLA complexes using centrifugal filtration cartridges (Vivaspin 500, 10 kDa) according to the manufacturer's protocol. The cartridges were additionally washed twice with 300 μl of 3% acetonitrile in 0.1% TFA. The peptides were dried in a Concentrator plus vacuum evaporator (Eppendorf) at 60°C for 6 hours and stored at -80°C until chromatograph mass spectrometry analysis.\n\nChromatographic mass spectrometry analysis of HLA I ligands.\nMass spectrometric analysis was performed on a Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). The chromatographic system was assembled using a pre-column-column scheme (column: sorbent - Aeris Peptide XB-C18 (Agilent, USA), length - 15 cm, internal diameter - 75 μm, particle size - 3 μm, pore diameter - 120 A, pre-column: sorbent - Aeris Peptide XB-C18, (Agilent, USA), length - 1 cm, internal diameter - 100 μm, particle size - 3 μm, pore diameter - 120 A, solvent A - 98.9% H2O, 1% MeOH, 0.1% formic acid; solvent B - 99.9% ACN, 0.1% formic acid), the chromatographic columns were thermostatted at 50°C. On the pre-column, application and washing were carried out for 10 minutes at a flow rate of 2 μl/min of solvent A. Separation was carried out at 300 nl/min, gradient: 1 min – 95% solvent A, 5% solvent B; The acetonitrile content was then increased linearly over 120 minutes to 60% solvent A and 40% solvent B. The chromatographic system was then washed with 95% solvent B for 10 minutes, followed by a 17-minute wash with 95% solvent A and 5% solvent B. Mass spectral acquisition mode: mass spectral acquisition time – 250 ms, mass/charge ratio range from 300 to 1250 m/z, criteria for preferential selection of ions for isolation and fragmentation – charge 2-5, the number of parent ions sampled in each cycle – no more than 50, minimum intensity – 100 counts/s. After the first analysis, the ion was forcibly excluded from the list of ions for fragmentation for 15 s. Fragmentation parameters: fragmentation spectra accumulation time – 100 ms, mass/charge ratio range from 100 to 2000 Th, voltage in the collision cell varied linearly from 25 V to 55 V, full cycle time – no more than 5.3 s.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "10.6019/PXD077095", "submissionType" : "COMPLETE", "publicationDate" : "2026-04-15", "updatedDate" : "2026-04-13", "submissionDate" : "2026-04-13", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Georgij Arapidi" ], "labPIs" : [ "Georgij Arapidi" ], "affiliations" : [ "Head of System Biology Lab. at Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency" ], "instruments" : [ "Q Exactive HF" ], "softwares" : [ ], "quantificationMethods" : [ "Spectrum counting" ], "sampleAttributes" : [ "ovary", "malignant neoplasm of ovary", "Homo sapiens (Human)", "malignant cell" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Ovary", "Malignant cell" ], "diseases" : [ "Malignant neoplasm of ovary" ], "references" : [ ], "experimentTypes" : [ "Immunopeptidomics", "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "P1037.raw", "P1027.mgf", "P1028.tandem.xml", "P1027.dat", "P1028.raw", "P1045.raw", "P1045.tandem.xml", "P1027.mzid", "P1028.mzid_P1028.MGF", "P1027.mzid_P1027.MGF", "checksum.txt", "P1037.mzid_P1037.MGF", "P1045.mzid_P1045.MGF", "P1028.mzid", "P1045.mgf", "P1037.dat", "P1037.tandem.xml", "P1045.mzid", "P1027.tandem.xml", "P1028.mgf", "P1027.raw", "P1037.mzid", "P1028.dat", "P1037.mgf", "P1045.dat" ], "highlights" : { } }, { "accession" : "PXD077041", "title" : "Adaptation of immunoaffinity protocol for the isolation of the cell line immunopeptides - immunopeptidomics part", "projectDescription" : "BACKGROUND: The peptide repertoire presented by Human Leukocyte Antigens (HLA) provides a snapshot of intracellular proteins, which is monitored by the immune system. Although mass spectrometry is the gold standard for identification of these peptides, the upstream steps of the immunopeptidomics workflow, including immunoprecipitation and peptides purification, vary and critically impact the peptide yield.\nAIM: To optimize conditions for the isolation of a larger number of HLA I ligands.\nMETHODS: Immunopeptidomes from cell lines were isolated using immunoaffinity followed by liquid chromatography-mass spectrometry analysis. Cell lines were lysed with various detergents (CHAPS, NP-40, SOD, Triton X-100) and eluted with TFA, urea, or thiocarbamate solutions.\nRESULTS: We optimized a protocol for immunoprecipitating peptide-HLA complexes and found that the number of identified peptides scales with the number of input cells, while being unaffected by the choice of lysis buffer.\nCONCLUSION: The optimized protocol allows the isolation of HLA I ligand peptides from suspension cell lines.", "dataProcessingProtocol" : "Raw data files acquired on the Q Exactive HF-X were converted to Mascot generic format (mgf) peak lists using MSConvert (ProteoWizard Software Foundation) with the following parameters: “--mgf --filter peakPicking true [1,2]”. The resulting peak lists were searched against the UniProt Knowledgebase (taxon human) concatenated with a reversed decoy set using MASCOT (version 2.5.1, Matrix Science Ltd., UK) and X! Tandem (ALANINE, 2017.02.01, The Global Proteome Machine Organization). The precursor and fragment mass tolerance were set at 20 ppm and 50 ppm, respectively. The database search parameters included no enzyme or possible post-translational modification (PTM). For X! Tandem, additional parameters were selected to check for protein N-terminal residue acetylation, peptide N-terminal glutamine ammonia loss, or peptide N-terminal glutamic acid water loss.", "sampleProcessingProtocol" : "Cell lines.\nThe following cell lines were used in this study: Jurkat (TIB-152, ATC, USA), Raji (CCL-86, ATCC), K562 (CCL-243, ATCC), and MDA-MB-231 (CRM-HTB-26, ATCC). Jurkat, Raji, and K562 suspension cell lines were cultured in RPMI 1640 medium (C330p, PanEco, Russia) containing 10% FBS (26140079, Gibco, USA), 50 U/ml gentamicin (Dalkhimfarm, Russia), and 1.6 mM glutamine GlutaMAX (35050061, Gibco). The adherent cell line MDA-MB 231 was cultured in DMEM (C420p, PanEco), 10% FBS, 50 U/ml gentamicin, and 1.6 mM glutamine. To detach the adhesion cell culture, 0.05% trypsin (15050057, Gibco) in Versene (P080p, PanEco) was used.\nAll cell lines were cultured to the required concentration, then centrifuged for 5 min at 150 g (Eppendorf 5804 R, USA). The cell pellet was washed three times with Dulbecco's phosphate buffered saline, pH 7.4 (PanEco). The supernatant was collected, and the cell pellet was stored at -80°C until further experiments.\nTo obtain antibodies to HLA I, a cell culture of the HB 95 hybridoma (producer of HLA-ABC w6/32 antibodies, ATCC) was cultivated. Primary cultivation was carried out in DMEM F12 Advanced medium (12634010, Gibco) with the addition of 10% FBS (inactivation at 56℃ for 30 minutes), 50 units/ml gentamicin, 1.6 mM glutamine. When the density of 600 thousand cells/ml was reached, the medium was changed to one containing half as much FBS. Thus, the concentration of FBS in the medium was gradually reduced. When the FBS concentration reached 0.5%, the medium was replaced with HyClone ADCF-mab (Cytiva, USA), 50 units/ml gentamicin, 1.6 mM glutamine. Cultivation in this medium until 80-90% of the cells died, then the supernatant containing antibodies was collected. Antibody concentrations in the cell medium were measured using the Easy-Titer mouse IgG Assay (23300, Thermo Scientific, USA) according to the manufacturer's recommendations.\nAll cells were regularly tested for mycoplasma contamination and cultured at 37°C and 5% CO2.\n\nPreparation of cell lysates.\nOne of the following lysis buffers was added to the frozen-dried cell pellet on ice:\nLys 1 – 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4) with cOmplet Protease Inhibitor Cocktail (11697498001, Roche, Switzerland).\nLys 2 – 10 mM CHAPS/PBS with cOmplet Protease Inhibitor Cocktail. Sonication was also performed (150 W at 50% pulse duration for 3 minutes on ice).\nLys 3 – 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, cOmplet Protease Inhibitor Cocktail, 1 mM PMSF, 1% octyl-D-glucopyranoside in PBS.\nLys 4 – 0.2 mM iodoacetamide, 1 mM EDTA, cOmplet Protease Inhibitor Cocktail, 1 mM PMSF, 1% octyl-β-D-glucopyranoside in PBS.\nThe cells were lysed for 1 hour on a rotator at +4 °C, then centrifuged for 15 minutes at 14,000 g and the supernatant was collected for further work.\n\nImmunoprecipitation of HLA complexes using magnetic beads.\nFor HLA I immunoprecipitation, 125 μl of Pierce Protein A/G Magnetic Beads (88803, Thermo Scientific) were used per 1 ml of lysate. Immunoprecipitation was performed according to the manufacturer's protocol. For one HLA I extraction, 125 μl of magnetic beads and 50 μg of w6/32 antibodies (MA1-80123, Invitrogen) were used. An aliquot of the magnetic beads was placed in 175 μl of wash buffer (2.7 mM KCl, 0.01 M Na2HPO4, 1.8 mM KH2PO4, 0.15 M NaCl, 0.05% Tween-20), then the magnetic beads were washed twice with 1 ml of wash buffer. w6/32 antibodies were added to the magnetic particle pellet and incubated for 1 hour. Then, 1 ml of clarified cell lysate was incubated with magnetic particles coated with specific antibodies for 1 hour at RT. After incubation, the particles were washed five times with phosphate-buffered saline (PBST), pH 7.4, containing 0.05% Tween-20. Precipitated peptide-HLA I complexes were eluted from the magnetic particles with 250 μl of 10% acetic acid. Peptides were purified by reverse-phase chromatography.\n\nPeptide purification.\nIsolated peptides were separated from HLA and desalted using an Extraction Manifold (Waters, USA) on Discovery DSC-18 columns (1 ml Tubes 100 mg, Supelco). The sorbent was activated with 100% methanol (MeOH), after which the columns were conditioned with a double volume of 1% MeOH in 0.1% trifluoroacetic acid (TFA). The sample was then loaded and washed with 2 ml of 1% MeOH in 0.1% TFA. Elution was carried out with 50% MeOH in 0.1% TFA. Purified peptides were dried in a Concentrator plus centrifugal concentrator (Eppendorf, Germany) and frozen at -80°C until LC-MS/MS analysis. Immediately prior to LC-MS/MS analysis, samples were dissolved in 15 µL of 1% MeOH in 0.1% TFA.\n\nChromatographic mass spectrometric analysis of HLA I ligand peptides.\nMass spectrometric analysis was performed on a Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). The chromatographic system was assembled using a pre-column-column scheme (column: sorbent - Aeris Peptide XB-C18 (Agilent, USA), length - 15 cm, internal diameter - 75 μm, particle size - 3 μm, pore diameter - 120 A, pre-column: sorbent - Aeris Peptide XB-C18, (Agilent, USA), length - 1 cm, internal diameter - 100 μm, particle size - 3 μm, pore diameter - 120 A, solvent A - 98.9% H2O, 1% MeOH, 0.1% formic acid; solvent B - 99.9% ACN, 0.1% formic acid), the chromatographic columns were thermostatted at 50°C. On the pre-column, application and washing were carried out for 10 minutes at a flow rate of 2 μl/min of solvent A. Separation was carried out at 300 nl/min, gradient: 1 min – 95% solvent A, 5% solvent B; The acetonitrile content was then increased linearly over 120 minutes to 60% solvent A and 40% solvent B. The chromatographic system was then washed with 95% solvent B for 10 minutes, followed by a 17-minute wash with 95% solvent A and 5% solvent B. Mass spectral acquisition mode: mass spectral acquisition time – 250 ms, mass/charge ratio range from 300 to 1250 m/z, criteria for preferential selection of ions for isolation and fragmentation – charge 2-5, the number of parent ions sampled in each cycle – no more than 50, minimum intensity – 100 counts/s. After the first analysis, the ion was forcibly excluded from the list of ions for fragmentation for 15 s. Fragmentation parameters: fragmentation spectra accumulation time – 100 ms, mass/charge ratio range from 100 to 2000 Th, voltage in the collision cell varied linearly from 25 V to 55 V, full cycle time – no more than 5.3 s.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "10.6019/PXD077041", "submissionType" : "COMPLETE", "publicationDate" : "2026-04-15", "updatedDate" : "2026-04-11", "submissionDate" : "2026-04-11", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Georgij Arapidi" ], "labPIs" : [ "Georgij Arapidi" ], "affiliations" : [ "Head of System Biology Lab. at Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency" ], "instruments" : [ "Q Exactive HF" ], "softwares" : [ ], "quantificationMethods" : [ "Peptide counting" ], "sampleAttributes" : [ "cell culture", "Homo sapiens (Human)" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Cell culture" ], "diseases" : [ 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(ac4C) reprograms R-loops and promotes cancer stem cell growth", "projectDescription" : "R-loop remodeling dynamically regulates chromatin states and gene expression; however, its exploitation by cancer to sustain self-renewal and malignancy remains poorly understood. Here, we found that glioblastoma (GBM) stem cells (GSCs) display highly active R-loops compared to differentiated tumor progeny and neural stem cells (NSCs). Genome-wide mapping by R-loop RNA chromatin immunoprecipitation sequencing (RR-ChIP-seq) revealed cell-specific enrichment and spatial accumulation of R-loops at promoter-proximal regions in GSCs, correlating with active transcription and open chromatin states. We profiled R-loop interactomes and identified N-Acetyltransferase 10 (NAT10), an RNA N4-acetylcytidine (ac4C)-modifying enzyme, as a high-affinity R-loop-binding protein in GSCs. Driven by transcriptional activation via OLIG1, NAT10 was overexpressed in GSCs. ac4C-specific RNA immunoprecipitation sequencing (ac4C-RIP-seq) on R-loop RNA revealed genome-wide mapping of ac4C-modified R-loops with abundant ac4C-modified R-loops that regulated the genome. NAT10 catalyzed widespread ac4C deposition on the RNA stand of R-loops, stabilizing promoter-associated R-loops, and facilitating open chromatin to sustain self-renewal through core stemness regulators, including transcription factor EGR1. NAT10 knockdown suppressed proliferation and maintenance in vitro and attenuated tumor growth in vivo. Pharmacological inhibition of NAT10/ac4C-modified R-loops using the small-molecule inhibitor remodelin phenocopied NAT10 genetic targeting, demonstrating therapeutic promise for targeting cancer.", "dataProcessingProtocol" : "IM/MS raw files were processed using MSFragger (v3.6). MS/MS spectra were searched against the OpenProt RNA transcriptome database consisting of alternative protein sequences resulting from all RNA transcripts reported by both Ensembl and NCBI RefSeq (https://openprot.org). The search parameters allowed for a 10ppm precursor ion mass error, 20ppm on fragment ion masses and a maximum 2 missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification. Oxidization of methionine acetylation of protein N-termini was set as variable modifications. False discovery rate (FDR) was capped at 0.05% prior to data export of raw peak areas. Peptide intensity was listed in Table S2. Differential analysis was performed in R using the DEP package (v1.30.0) based on peptide intensity. Proteins were identified as differentially associated with R-loops using a cutoff of adjusted P value < 0.05 and |log₂ fold change| ≥ 1.", "sampleProcessingProtocol" : "For S9.6 IP followed by mass spectrometry analysis, a total of 1 × 10⁷ non-crosslinked GSC, DGC, and NSC cells were collected and lysed on ice for 10 minutes in cell lysis buffer (85 mM KCl, 5 mM PIPES (pH 8.0), and 0.5% NP-40) supplemented with protease inhibitors. Nuclear pellets were resuspended in nuclear lysis buffer (10 mM Tris-HCl pH 7.5, 200 mM NaCl, 2.5 mM MgCl₂, 0.2% sodium deoxycholate, 0.1% SDS, 0.05% sodium lauroyl sarcosinate, and 0.5% Triton X-100) supplemented with protease inhibitors, then sonicated for 5 minutes to shear chromatin using 30% of maximum output power (30 seconds on, 30 seconds off). The resulting extracts were diluted in R-loop IP buffer (10 mM Tris-HCl pH 7.5, 200 mM NaCl, 2.5 mM MgCl₂, 0.5% Triton X-100, 0.05% sodium deoxycholate, 0.025% SDS, 0.0125% sodium lauroyl sarcosinate) supplemented with and inhibitors, and subsequently incubated with the S9.6 antibody conjugated protein A/G magnetic beads for immunoprecipitation at 4℃ for 30 minutes with rotation. For dRNH1 IP, lysates were incubated with His-tagged dRNH1 probe and a subsequent anti-His antibody conjugated protein A/G magnetic beads. Mouse IgG served as a negative control. Each experiment was added with 12 μl of freshly prepared 0.1μg/μl RNase A and incubated at 4℃ for 1 hour 30 minutes with rotation for to remove the potential contamination of free single strand RNA. After precipitation, beads were washed four times with RSBT buffer (10 mM Tris-HCl pH 7.5, 200 mM NaCl, 2.5 mM MgCl₂, 0.5% Triton X-100) and twice with RSB buffer (10 mM Tris-HCl pH 7.5, 200 mM NaCl, 2.5 mM MgCl₂) to remove non-specific binding. For mass spectrometry, proteins were eluted in 5% SDS buffer and subjected to S-Trap trypsin digestion. The resultant tryptic peptides were extracted with 70% ACN/5% formic acid (FA), vacuum dried, and re-constituted in 20 μl 3%ACN/0.1% FA. LC-MS/MS analysis was carried out using a nanoElute2 UHPLC system (Bruker Daltonics, Bremen, Germany) coupled to the timsTOF Pro2 mass spectrometer mass spectrometer (Bruker Daltonics), using a CaptiveSpray nanoelectrospray (Bruker Daltonics). 2µL, roughly 200 ng, of peptide digest was loaded on a capillary C18 column (25 cm length, 75 μm inner diameter, 1.6 μm particle size, 120 Å pore size; IonOpticks Aurora Gen3, Fitzroy, VIC, AUS). Peptides were separated at 55°C using a 60 min gradient at a flow rate of 300 nL/min (mobile phase A (MPA): 0.1% FA; mobile phase B (MPB): 0.1% FA in acetonitrile). A linear gradient of 2–35% MPB was applied for 60 min, followed by a 5 min wash at 95% MFB before equilibrating the column at 2% MFB for 6 min. The Bruker timsTOF Pro2 was operated in data-dependent PASEF mode collecting full scan mass spectra from 100 and 1700 m/z. Ion mobility resolution was set to 0.60–1.60 V·s/cm over a ramp time of 100 ms. Data-dependent acquisition was performed using 10 PASEF MS/MS scans per 1.1 second cycle. Active exclusion time window was set to 0.4 min, and the intensity threshold for MS/MS fragmentation was et to 2.5e4 while low m/z and singly charged ions were excluded from PASEF precursor selection. MS/MS spectra were acquired via ramped collision energy as function of ion mobility.", "projectTags" : [ ], "keywords" : [ "Glioblastoma stem cell", "Glioblastoma", "Cancer stem cell", "Nat10", "R-loop remodeling", "Ac4c" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-10", "updatedDate" : "2026-04-10", "submissionDate" : "2026-04-10", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Weichi Wu" ], "labPIs" : [ "Jeremy Naftali Rich" ], "affiliations" : [ "University of North Carolina, Chapel Hill" ], "instruments" : [ "autoflex" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "glioblastoma", "stem cell", "cell culture", "Homo sapiens (Human)", "permanent cell line cell", "cell suspension culture" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Cell culture", "Cell suspension culture", "Stem cell", "Permanent cell line cell" ], "diseases" : [ "Glioblastoma" ], "references" : [ ], "experimentTypes" : [ "Affinity purification coupled with mass spectrometry proteomics" ], "sdrf" : "", "projectFileNames" : [ "IgG-R2-112223_Rich_Po_S8_Slot2-8_1_2292.d.zip", "Rloop_Mass_spec.xlsx", "DGC23-R1-112223_Rich_Po_S3_Slot2-3_1_2291.d.zip", "checksum.txt", "DGC23-R2-112223_Rich_Po_S4_Slot2-4_1_2290.d.zip", "GSC23-R2-112223_Rich_Po_S2_Slot2-2_1_2289.d.zip", "NSC11-R2-112223_Rich_Po_S6_Slot2-6_1_2296.d.zip", "NSC11-R1-112223_Rich_Po_S5_Slot2-5_1_2295.d.zip", "IgG-R1-112223_Rich_Po_S7_Slot2-7_1_2294.d.zip", "GSC23-R1-112223_Rich_Po_S1_Slot2-1_1_2293.d.zip" ], "highlights" : { } }, { "accession" : "PXD076837", "title" : "Phosphoproteomics of tau from tau knockout mice expressing tau or phospho-deficient tauT205A in active hippocampal cells upon fear conditioning", "projectDescription" : "This dataset describes the mass spectrometric identification of tau phosphorylation sites in hippocampal neurons active during fear memory encoding. Tau knockout (tau‑/‑) mice were used as a background to selectively express human 2N4R tau variants in behaviourally activated neuronal populations. Tau‑/‑ mice received bilateral injections into the dorsal and ventral hippocampus with doxycycline‑regulated AAV vectors under control of the robust activity‑marking (RAM) promoter. Experimental groups expressed either wild‑type human 2N4R tau (tauWT; n = 4), a phosphorylation‑deficient tau mutant (tauT205A; n = 4), or eGFP as a negative control (n = 2). Following postoperative recovery, mice underwent fear conditioning. Hippocampal tissue was collected 120 minutes after conditioning, prior to induction of transgene expression. Temporal control of tau, tauT205A, or eGFP expression was achieved by doxycycline withdrawal, allowing expression during a defined 20‑hour window corresponding to neuronal activity associated with fear memory encoding. Tau protein was subsequently immunoprecipitated from hippocampal lysates and subjected to bottom‑up phosphoproteomic analysis. Immunoprecipitated tau was reduced, alkylated, and digested with trypsin, followed by enrichment of phosphopeptides using titanium dioxide (TiO₂) affinity chromatography. Enriched phosphopeptides were buffer‑exchanged, dried by vacuum centrifugation, resuspended, and analysed by liquid chromatography–tandem mass spectrometry (LC‑MS/MS). This dataset enables comparative analysis of tau phosphorylation patterns associated with neuronal activation during fear learning and provides insight into the role of the T205 phosphorylation site in activity‑dependent tau regulation.", "dataProcessingProtocol" : "Peak lists were generated using MASCOT Distiller and searched with the MASCOT search engine (versions v3.0.0 and v3.1.0; Matrix Science). Searches were performed against the Swiss‑Prot protein sequence database (release 19 January 2024; 570,420 sequences; 206,321,560 residues) restricted to Mammalia taxonomy. Database searches were conducted using the MS/MS ion search type with semi‑tryptic enzyme specificity, allowing up to three missed cleavages. Carbamidomethylation of cysteine residues was specified as a fixed modification, while acetylation of asparagine, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine residues were included as variable modifications. Precursor ion mass tolerance was set to ±5 ppm and fragment ion mass tolerance to ±0.05 Da. The default Mascot minimum peptide length (7 amino acid residues) was applied.", "sampleProcessingProtocol" : "Tau protein was immunoprecipitated from hippocampal tissue extracts adjusted to a total protein concentration of 6 µg/µl. Extracts were incubated with 60 μl Pierce Anti-HA Magnetic Beads (no. 88836, Thermo Scientific) for 4 h at 4 °C with gentle rotation. Beads were washed three times with RIPA buffer for 5 min at 4 °C, followed by one rinse and one wash with 20 mM Tris‑HCl (pH 7.4), 2 mM CaCl₂, and 150 mM NaCl. To eliminate residual detergent and protease inhibitors, beads were transferred to a new tube and washed twice with the same Tris‑based buffer, followed by three washes with freshly prepared 20 mM ammonium bicarbonate containing 150 mM NaCl. Beads were then resuspended in 1× trypsin digestion buffer (V5111, Promega). On‑bead proteins were reduced with 1 mM tris(2‑carboxyethyl)phosphine (TCEP) for 10 min at 56 °C and alkylated with 10 mM iodoacetamide (I1149, Sigma‑Aldrich) for 30 min at ambient temperature in the dark. Proteolytic digestion was performed on‑bead using sequencing‑grade trypsin (V5111, Promega) at a 1:25 (w/w) enzyme‑to‑protein ratio for 16 h at 37 °C, supplemented with ProteaseMAX surfactant (V2072, Promega). Phosphopeptides were enriched using TiO₂ spin tips (GL Sciences) according to the manufacturer’s instructions. Trifluoroacetic acid (TFA) was added to the enriched samples to a final concentration of 0.1%. Peptides were desalted using tC18 cartridges (WAT036805, Waters), which were conditioned sequentially with 100% acetonitrile, 50% acetonitrile containing 0.5% acetic acid, and 0.1% TFA. After sample loading, cartridges were washed with 0.1% TFA and 0.5% acetic acid, and phosphopeptides were eluted with 80% acetonitrile containing 0.5% acetic acid. Eluted peptides were dried by vacuum centrifugation (Eppendorf) for 45 min at 45 °C and resuspended in 0.2% heptafluorobutyric acid containing 1% formic acid. Samples were stored at –80 °C until analysis. Phosphopeptides were analysed by LC‑MS/MS using an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) equipped with a FAIMS Pro Duo ion source and coupled to a Vanquish Neo UHPLC system (Thermo Electron). Peptides (3–6 µg injected in 1–7 µl) were first captured on a PepMap Neo C18 trap cartridge (300 µm, 5 µm; Thermo Scientific) and separated on a C18 reverse‑phase analytical column (~25 cm; ReproSil‑Pur, 1.9 µm, 200 Å; Dr. Maisch GmbH) housed in a column oven maintained at 45 °C (Sonation). Peptides were eluted over a 40‑min linear water–acetonitrile gradient containing 0.1% formic acid at a flow rate of 200 nl/min.", "projectTags" : [ ], "keywords" : [ "Phosphoproteomics", "Tau", "Engram", "Hippocampus", "Fear conditioning" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-10", "updatedDate" : "2026-04-10", "submissionDate" : "2026-04-10", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Arne Ittner" ], "labPIs" : [ "Arne Ittner" ], "affiliations" : [ "Flinders Health and Medical Research Institute, College of Medicine and Public Health, Flinders University, Adelaide, Australia" ], "instruments" : [ "4800 Plus MALDI TOF/TOF" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "brain", "Mus musculus (Mouse)" ], "organisms" : [ "Homo sapiens (human)", "Mus musculus (mouse)" ], "organismsPart" : [ "Brain" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "F015694.dat", "F015670.dat", "AP310125_AI_1_20250214222023.raw", "AP310125_AI_13.raw", "F015680.dat", "AP310125_AI_5_20250215055016.raw", "F015691.dat", "F015667.dat", "AP310125_AI_7.raw", "F015678.dat", "AP310125_AI_15.raw", "AP310125_AI_2_20250215001252.raw", "AP310125_AI_4_20250215035802.raw", "F015676.dat", "AP310125_AI_6.raw", "checksum.txt", "F015693.dat", "F015695.dat", "F015673.dat", "AP310125_AI_12.raw", "AP310125_AI_14.raw" ], "highlights" : { } }, { "accession" : "PXD076866", "title" : "Adaptation of immunoaffinity protocol for the isolation of the cell line immunopeptides - proteomics part", "projectDescription" : "BACKGROUND: The peptide repertoire presented by Human Leukocyte Antigens (HLA) provides a snapshot of intracellular proteins, which is monitored by the immune system. Although mass spectrometry is the gold standard for identification of these peptides, the upstream steps of the immunopeptidomics workflow, including immunoprecipitation and peptides purification, vary and critically impact the peptide yield.\nAIM: To optimize conditions for the isolation of a larger number of HLA I ligands.\nMETHODS: Immunopeptidomes from cell lines were isolated using immunoaffinity followed by liquid chromatography-mass spectrometry analysis. Cell lines were lysed with various detergents (CHAPS, NP-40, SOD, Triton X-100) and eluted with TFA, urea, or thiocarbamate solutions.\nRESULTS: We optimized a protocol for immunoprecipitating peptide-HLA complexes and found that the number of identified peptides scales with the number of input cells, while being unaffected by the choice of lysis buffer.\nCONCLUSION: The optimized protocol allows the isolation of HLA I ligand peptides from suspension cell lines.", "dataProcessingProtocol" : "Raw data files acquired on the Q Exactive HF-X were converted to Mascot generic format (mgf) peak lists using MSConvert (ProteoWizard Software Foundation) with the following parameters: “--mgf --filter peakPicking true [1,2]”. The resulting peak lists were searched against the UniProt Knowledgebase (taxon human) concatenated with a reversed decoy set using MASCOT (version 2.5.1, Matrix Science Ltd., UK) and X! Tandem (ALANINE, 2017.02.01, The Global Proteome Machine Organization). The precursor and fragment mass tolerance were set at 20 ppm and 50 ppm, respectively. Database search parameters included the following: tryptic digestion with 1 possible missed cleavage; static modification for carbamidomethyl (C); dynamic/flexible modifications for oxidation (M). For X! Tandem, additional parameters were selected to check for protein N-terminal residue acetylation, peptide N-terminal glutamine ammonia loss, or peptide N-terminal glutamic acid water loss.", "sampleProcessingProtocol" : "Cell lines.\nThe following cell lines were used in this study: Jurkat (TIB-152, ATC, USA), Raji (CCL-86, ATCC), K562 (CCL-243, ATCC), and MDA-MB-231 (CRM-HTB-26, ATCC). Jurkat, Raji, and K562 suspension cell lines were cultured in RPMI 1640 medium (C330p, PanEco, Russia) containing 10% FBS (26140079, Gibco, USA), 50 U/ml gentamicin (Dalkhimfarm, Russia), and 1.6 mM glutamine GlutaMAX (35050061, Gibco). The adherent cell line MDA-MB 231 was cultured in DMEM (C420p, PanEco), 10% FBS, 50 U/ml gentamicin, and 1.6 mM glutamine. To detach the adhesion cell culture, 0.05% trypsin (15050057, Gibco) in Versene (P080p, PanEco) was used.\nAll cell lines were cultured to the required concentration, then centrifuged for 5 min at 150 g (Eppendorf 5804 R, USA). The cell pellet was washed three times with Dulbecco's phosphate buffered saline, pH 7.4 (PanEco). The supernatant was collected, and the cell pellet was stored at -80°C until further experiments.\nTo obtain antibodies to HLA I, a cell culture of the HB 95 hybridoma (producer of HLA-ABC w6/32 antibodies, ATCC) was cultivated. Primary cultivation was carried out in DMEM F12 Advanced medium (12634010, Gibco) with the addition of 10% FBS (inactivation at 56℃ for 30 minutes), 50 units/ml gentamicin, 1.6 mM glutamine. When the density of 600 thousand cells/ml was reached, the medium was changed to one containing half as much FBS. Thus, the concentration of FBS in the medium was gradually reduced. When the FBS concentration reached 0.5%, the medium was replaced with HyClone ADCF-mab (Cytiva, USA), 50 units/ml gentamicin, 1.6 mM glutamine. Cultivation in this medium until 80-90% of the cells died, then the supernatant containing antibodies was collected. Antibody concentrations in the cell medium were measured using the Easy-Titer mouse IgG Assay (23300, Thermo Scientific, USA) according to the manufacturer's recommendations.\nAll cells were regularly tested for mycoplasma contamination and cultured at 37°C and 5% CO2.\n\nPreparation of cell lysates.\nOne of the following lysis buffers was added to the frozen-dried cell pellet on ice:\nLys 1 – 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4) with cOmplet Protease Inhibitor Cocktail (11697498001, Roche, Switzerland).\nLys 2 – 10 mM CHAPS/PBS with cOmplet Protease Inhibitor Cocktail. Sonication was also performed (150 W at 50% pulse duration for 3 minutes on ice).\nLys 3 – 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, cOmplet Protease Inhibitor Cocktail, 1 mM PMSF, 1% octyl-D-glucopyranoside in PBS.\nLys 4 – 0.2 mM iodoacetamide, 1 mM EDTA, cOmplet Protease Inhibitor Cocktail, 1 mM PMSF, 1% octyl-β-D-glucopyranoside in PBS.\nThe cells were lysed for 1 hour on a rotator at +4 °C, then centrifuged for 15 minutes at 14,000 g and the supernatant was collected for further work.\n\nImmunoprecipitation of HLA complexes using magnetic beads.\nFor HLA I immunoprecipitation, 125 μl of Pierce Protein A/G Magnetic Beads (88803, Thermo Scientific) were used per 1 ml of lysate. Immunoprecipitation was performed according to the manufacturer's protocol. For one HLA I extraction, 125 μl of magnetic beads and 50 μg of w6/32 antibodies (MA1-80123, Invitrogen) were used. An aliquot of the magnetic beads was placed in 175 μl of wash buffer (2.7 mM KCl, 0.01 M Na2HPO4, 1.8 mM KH2PO4, 0.15 M NaCl, 0.05% Tween-20), then the magnetic beads were washed twice with 1 ml of wash buffer. w6/32 antibodies were added to the magnetic particle pellet and incubated for 1 hour. Then, 1 ml of clarified cell lysate was incubated with magnetic particles coated with specific antibodies for 1 hour at RT. After incubation, the particles were washed five times with phosphate-buffered saline (PBST), pH 7.4, containing 0.05% Tween-20. Precipitated peptide-HLA I complexes were eluted from the magnetic particles with 250 μl of 10% acetic acid. Peptides were purified by reverse-phase chromatography.\n\nPreparing eluates for proteomic analysis.\nTo reduce disulfide bonds in the eluates containing HLA complexes and their peptide ligands, as well as antibodies, dithiothreitol (DTT) was added to a final concentration of 5 mM and incubated for 30 minutes at room temperature (RT). Iodoacetamide was then added to a final concentration of 10 mM, and the samples were incubated in the dark at RT for 20 minutes. The samples were then diluted 5-fold with 50 mM ammonium bicarbonate solution. Trypsin (Sequencing Grade Modified Trypsin, Promega, USA) was added to each sample at a ratio of 1:100 (m/m) and incubated for 14 hours at 37°C. The reaction was stopped by adding formic acid to a final concentration of 5%. Tryptic peptides were desalted using Empore SDB-RPS CDS (Fisher Scientific) according to the manufacturer's protocol. Purified peptides were dried in a vacuum centrifuge and stored at -80°C until LC-MS/MS analysis.\n\nChromatographic mass spectrometric analysis of tryptic peptides.\nDried tryptic peptides were dissolved in 3% ACN and 0.1% TFA and applied to a column (75 μm diameter, 50 cm length) with Aeris Peptide XB-C18 2.6 μm sorbent (Phenomenex). Peptides were separated on an Ultimate 3000 Nano LC System (Thermo Fisher Scientific) coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) using a nanoelectrospray source (Thermo Fisher Scientific). Peptides were loaded onto a column thermostatted at 40°C in buffer A (0.2% formic acid (FA) in water) and eluted with a linear (120 min) gradient of 4>55% buffer B (0.1% FA, 19.9% ​​water, 80% acetonitrile) in A at a flow rate of 350 nL/min. Before each new load, the column was washed with 95% buffer B in A for 5 min and equilibrated with buffer A for 5 min. Mass spectrometric data were stored with automatic switching between MS1 ​​scans and up to 15 MS/MS scans (topN method). The target value for the MS1 scan was set to 3 × 106 in the range of 300-1200 m/z with a maximum ion injection time of 60 ms and a resolution of 60,000. Precursor ions were isolated with a window width of 1.4 m/z and a fixed first mass of 100.0 m/z. Precursor ions were fragmented using high-energy dissociation in a C-trap with a normalized collision energy of 28 eV. MS/MS scans were stored at a resolution of 15,000 at m/z 400 and at a resolution of 100,000 for target ions in the range of 200-2000 m/z with a maximum ion injection time of 30 ms.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "10.6019/PXD076866", "submissionType" : "COMPLETE", "publicationDate" : "2026-04-15", "updatedDate" : "2026-04-10", "submissionDate" : "2026-04-10", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Georgij Arapidi" ], "labPIs" : [ "Georgij Arapidi" ], "affiliations" : [ "Head of System Biology Lab. at Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency" ], "instruments" : [ "Q Exactive HF" ], "softwares" : [ ], "quantificationMethods" : [ "Spectrum counting" ], "sampleAttributes" : [ "cell culture", "Homo sapiens (Human)" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Cell culture" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : 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"P291.mgf" ], "highlights" : { } }, { "accession" : "PXD076861", "title" : "Native MS and intact-protein MS datasets for NreB and NreB_PAS supporting the study “A labile sulfur ligand in a three-cysteine-coordinated [2Fe-2S] cluster mediates sulfide sensing in NreB”", "projectDescription" : "This ProteomeXchange dataset contains native mass spectrometry and intact-protein mass spectrometry data generated in support of the manuscript “A labile sulfur ligand in a three-cysteine-coordinated [2Fe-2S] cluster mediates sulfide sensing in NreB”. The dataset includes intact-protein MS measurements of purified His_NreB and His_NreB_PAS for molecular-mass verification, as well as native MS measurements of NreB_PAS under untreated, GSH-treated, GSH plus NaHS-treated, and pH-dependent conditions. These data were used to characterize Fe-S cluster-associated adduct states and to compare the distributions of species detected under different conditions. Raw data acquired on a Bruker Impact HD mass spectrometer and the corresponding deconvolution outputs generated using Bruker DataAnalysis 4.2 are included together with a deconvolution summary table.", "dataProcessingProtocol" : "Mass spectrometric analysis was performed on a Bruker Impact HD high-resolution Q-TOF mass spectrometer equipped with an Ultimate3000 system. Samples were directly infused into the positive-ion ESI source at a flow rate of 0.3 mL/h, and spectra were acquired over an m/z range of 900-4500. Instrument calibration was performed using an electrospray calibrant solution. Data acquisition was carried out using OtofControl version 3.4, and spectral analysis and deconvolution were performed using Compass DataAnalysis version 4.2 (Bruker Daltonik). For each raw .d file, the corresponding deconvolution output is provided. For quantitative comparison of native MS species, the intensity of each peak type was normalized to the summed intensity of all six detected peak types. No peptide-spectrum matching or database searching was performed, because the dataset contains intact-protein and native MS measurements rather than bottom-up proteomics data.", "sampleProcessingProtocol" : "Purified recombinant His-tagged NreB and His-tagged NreB_PAS proteins were used for intact-protein MS measurements for molecular-mass verification. For native MS experiments, purified NreB_PAS samples were prepared in 100 mM Tris-HCl buffer containing 100 mM NaCl, 5 mM KCl, 5 mM MgCl2, and 5% (v/v) glycerol. Standard reaction samples were prepared at pH 8.0 using 80 μM protein, which was incubated with 5 mM GSH for 10 min, followed by addition of NaHS at a 10-fold molar excess relative to the protein concentration and further incubation for 4 min under aerobic conditions. The reacted samples were then transferred into an anaerobic glovebox and buffer-exchanged into 200 mM ammonium acetate, pH 8.0, using 10 kDa cutoff centrifugal filters to remove excess GSH and NaHS, and the final protein concentration was adjusted to 80 μM. Additional untreated samples were prepared by directly buffer-exchanging purified NreB_PAS into 200 mM ammonium acetate at either pH 7.0 or pH 8.0. For pH 7.0 treatment experiments, purified NreB_PAS samples were prepared in 100 mM Tris-HCl buffer, pH 7.0, containing 100 mM NaCl, 5 mM KCl, 5 mM MgCl2, and 5% (v/v) glycerol, treated with NaHS, and then transferred under anaerobic conditions into 200 mM ammonium acetate, pH 7.0, using the same buffer-exchange procedure described above. No enzymatic digestion, peptide enrichment, or chromatographic fractionation was performed.", "projectTags" : [ ], "keywords" : [ "[2fe-2s]", "Nreb", "Sulfide sensing", "Native ms", "Intact protein ms", "Nreb_pas", "Fe-s cluster", "Nahs", "Gsh", "Bruker impact hd" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-15", "updatedDate" : "2026-04-10", "submissionDate" : "2026-04-10", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "chao tang" ], "labPIs" : [ "Yongzhen Xia" ], "affiliations" : [ "State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, People’s Republic of China" ], "instruments" : [ "impact" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Bacillus licheniformis DSM 13 = ATCC 14580" ], "organisms" : [ "Bacillus licheniformis dsm 13 = atcc 14580" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Top-down proteomics" ], "sdrf" : "", "projectFileNames" : [ "repeat_3_NreB_PAS_NaHS_in_pH7_RAW.d.zip", "repeat_2_NreB_PAS_in_pH7_RAW.d.zip", "NreB_PAS_DECONV.d.zip", "His_NreB_RAW.d.zip", "README_PRIDE_NreBPAS_NativeMS_IntactMS.txt.txt", "repeat_1_NreB_PAS_in_pH8_DECONV.d.zip", "checksum.txt", "repeat_2_NreB_PAS_NaHS_in_pH7_DECONV.d.zip", "repeat_3_NreB_PAS_in_pH7_RAW.d.zip", "repeat_1_NreB_PAS_in_pH8_RAW.d.zip", "repeat_1_NreB_PAS_pH7_RAW.d.zip", "repeat_1_NreB_PAS_NaHS_in_pH7_RAW.d.zip", "NreB_NreBPAS_Deconvolution_Summary_Table.xlsx.xlsx", "repeat_2_NreB_PAS_in_pH7_DECONV.d.zip", "NreB_PAS_GSH_NaHS_RAW.d.zip", "repeat_2_NreB_PAS_in_pH8_DECONV.d.zip", "His_NreB_PAS_RAW.d.zip", "repeat_3_NreB_PAS_NaHS_in_pH7_DECONV.d.zip", "NreB_PAS_GSH_DECONV.d.zip", "repeat_2_NreB_PAS_in_pH8_RAW.d.zip", "repeat_1_NreB_PAS_pH7_DECONV.d.zip", "repeat_2_NreB_PAS_NaHS_in_pH7_RAW.d.zip", "NreB_PAS_GSH_NaHS_DECONV.d.zip", "NreB_PAS_RAW.d.zip", "His_NreB_DECONV.d.zip", "repeat_3_NreB_PAS_in_pH7_DECONV.d.zip", "NreB_PAS_GSH_RAW.d.zip", "His_NreB_PAS_DECONV.d.zip", "repeat_1_NreB_PAS_NaHS_in_pH7_DECONV.d.zip", "repeat_3_NreB_PAS_in_pH8_DECONV.d.zip", "repeat_3_NreB_PAS_in_pH8_RAW.d.zip" ], "highlights" : { } }, { "accession" : "PXD076857", "title" : "Structure of Pex8 in complex with peroxisomal receptor Pex5 reveals its essential role in peroxisomal cargo translocationSMS", "projectDescription" : "Please provide an overall description of your study, think something similar in scope to the manuscript abstractPeroxisomes are essential cellular organelles that enable the sequestered execution of a broad range of metabolic processes. Due to the lack of an internal protein synthesis machinery, they entirely depend on the import of target proteins to carry out their functions within peroxisomes. While the process of cargo/receptor recognition is well understood, knowledge about the molecular mechanisms of the subsequent translocation steps, including cargo release and receptor recycling, is lacking behind. Here, we provide structural and functional evidence on the role of Pex8 in these processes. First, we show that Pex8 in yeast is essential for peroxisomal cargo translocation, irrespective of the mechanism of receptor/cargo recognition. Next, we reveal that Pex8 binds through an irregular twelvefold HEAT repeat array to a short three-helical bundle within the otherwise unfolded N-terminal domain of the Pex5 receptor. Impairing this interaction abolishes peroxisomal protein translocation. It is complemented by a secondary autonomous Pex8 cargo-like interaction site with the C-terminal domain of Pex5, thus generating a bipartite interaction between the two proteins. Our data support a model in which Pex5/Pex8 complex formation allows assembly with the peroxisomal Pex2/Pex10/Pex12 E3-ubiquitin ligase complex to initiate recycling of the receptor. In summary, our findings provide in-depth insight into the transition from cargo release into peroxisomes to receptor recycling, which is essential to uncover the overall process of the peroxisomal cargo translocation.", "dataProcessingProtocol" : "Please provide a couple of sentences on the bioinformatics pipeline used, main search parameters, quantitative analysis, software tools and versions included. Think something similar in scope to the Data Analysis section of your manuscriptCrosslinked peptides were identified using the software pLink2 (version 2.3.9). Searches were performed against the forward and reverse sequences of protein samples, including tryptic peptides 5-60 amino acids in length and allowing a maximum of three missed cleavages. Oxidation of methionine was set as variable and carbamidomethylating of cysteine was treated as a fixed modification. Lysine, tyrosine, serine, threonine and the N-terminus were set as one crosslinked site, and any other residue as second crosslinked site. The mass tolerance of the search of MS1 and MS2 spectra was set to 20 ppm, and identified MS1 spectra were subsequently filtered by a mass tolerance of 10 ppm and a false discovery rate (FDR) of 5% at peptide-spectrum match (PSM) level. pLink2 results were subsequently filtered by the following criteria: PSMs having E-values above 1 x 10-2 or comprising adjacent tryptic peptides of the same protein were rejected. Additionally, crosslinks covering the protein N-terminus were omitted. Only crosslinked residue pairs that were identified in 3 out 4 replicates were retained for further interpretation. The resulting crosslinking data were then visualized using XiNET (version 2.0.0).", "sampleProcessingProtocol" : "Please provide a short description on the sample preparation steps, separation, enrichment strategies and mass spectrometry protocols includedProtein samples were diluted to a final concentration of 21 µM, using crosslinking buffer that contained 50 mM HEPES (pH 7.5) and 150 mM NaCl at 10°C. For each crosslinking reaction 6 µL protein solution was mixed with a second solution of the same protein at the same concentration. After 10 min incubation time, 0.5 µL crosslinking reagent S-SDA (Thermo Fisher Scientific, #26173) dissolved in ultrapure water was added to the protein solution, to achieve a final molar excess of crosslinker over protein of 11.5. N-hydroxy succinimide ester (NHS)-based crosslinking was performed at RT for 30 min. Next, diazirine-based crosslinking was performed using an in-house build LED UV light source (∼ 30 x103 µW/cm2) for 20 min on ice. For separating crosslinking reaction products by SDS-PAGE, 3.125 µL of the five-times diluted sample buffer containing 10% [w/v] SDS, 25% [v/v] β-mercaptoethanol, 50% [v/v] glycerol, 1.25 M Tris/HCl (pH 6.8), and 0,01% [w/v] bromophenol blue was added and the mixture was incubated for 8 min at 95°C. Coomassie-stained protein bands were excised and destained by alternating incubations with 10 mM ammonium bicarbonate (AmBic) or 5 mM AmBic / 50% [v/v] ethanol for 10 min at room temperature (RT). Gel slices were dehydrated in 100% ethanol for 10 min at RT, followed by a reduction with 5 mM TCEP in 10 mM AmBic for 45 min at 56°C, and alkylation with 50 mM iodoacetamide (IAA), in 10 mM AmBic for 15 min at RT in the dark. Excess IAA was washed out using 10 mM AmBic and 5 mM AmBic / 50% [v/v] ethanol for 10 min at RT. For subsequent proteolytic digestion, gel slices were dried using a vacuum centrifuge at 30°C for 45 min. Trypsin (Promega) was added at a protease:protein mass ratio of 1:20 and the mixture was digested for 16 h at 37°C. Peptides were eluted using a solution of 50% (v/v) acetonitrile and 50% (v/v) ethanol, and sonicated in an ice-cooled water bath sonicator for 10 min. Eluted peptides were dried in a vacuum centrifuge and either stored at – 80°C or directly resuspended for desalting prior to the LC-MS analysis. For LC-MS analysis peptides were resuspended in 0.1% (v/v) trifluoroacetic acid and precipitates were pelleted by centrifugation for 6 min at 12,000 rpm at RT). Peptides were separated on a UltimateTM 3000 RSLCnano system (Thermo Fisher Scientific), equipped with a PepMapTM (C18,0.3 mm ID x 5 mm L) precolumn (Thermo Fisher Scientific) and an Acclaim PepMapTM (C18, 75 μm ID x 500 mm L, 100 Å pore size) analytical column (Thermo Fisher Scientific), using a binary solvent system consisting of 0.1% [v/v] formic acid (solvent A) and 86% [v/v] acetonitrile/0.1% [v/v] formic acid (solvent B) at 40°C. Peptides were loaded at 1% solvent B for 3 min at a flow rate of 10 µL x min-1 and were eluted by applying a three-step gradient, starting after 4 min with 2% solvent B at a flow rate of 0.3 µL x min-1 over 24% solvent B at 67 min, 31% at 80 min, and reaching 47% solvent B after 91 min. Subsequently, the column was flushed with 95% solvent B for 5 min. Finally, the column was equilibrated for 20 min at 1% solvent B with a flow rate of 0.35 µL x min-1. Separated peptides were measured on a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific), equipped with a nano electrospray ion source and a stainless steel emitter, using a data-dependent acquisition mode with a scan range of m/z 400 to 1,450 at a maximum resolution of 140,000 at m/z 400, an automatic gain control (AGC) of 3 x 106 ions and a maximum fill time of 50 ms for MS1 scans. The 10 most intense precursor ions with charge states between 2+ and 8+ were selected for higher-energy collisional dissociation (HCD) with stepped normalized collision energies of 24%, 28% and 30%. MS2 data were acquired within a scan range of m/z 200 to 2,000 at a resolution of 70,000, with an AGC of 5 x 104 and a maximum fill time of 120 ms. A dynamic exclusion time of 30 s was set for already fragmented precursor ions.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-17", "updatedDate" : "2026-04-10", "submissionDate" : "2026-04-10", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Julian Bender" ], "labPIs" : [ "Dr Bettina" ], "affiliations" : [ "Lab head's affiliation, such as: depChair of Biochemistry II, Theodor Boveri-Institute, Biocenter, University of Würzburg, Würzburg, Germanyartment, lab, institute and country" ], "instruments" : [ "Q Exactive Plus" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Komagataella phaffii" ], "organisms" : [ "Komagataella phaffii" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.1101/2025.08.30.673231" ], "experimentTypes" : [ "Data-dependent acquisition", "Chemical cross-linking coupled with mass spectrometry proteomics" ], "sdrf" : "Lessdrf v0.1.0 Komagataella phaffii Label free sample Proteomic profiling by mass spectrometry Q exactive plus Nt=xlink:sda; 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Yet, discrete intermediates and host factors supporting nucleic acid uncoating in susceptible cells are largely elusive. Using cryo-electron tomography (cryo-ET) and superresolution microscopy we show that human cells lacking the ubiquitin E3-ligase Mindbomb-1 (Mib1) deadlock adenovirus (AdV) particles at ~150nm radial distance from the nuclear pore complex (NPC). Cryo-ET revealed that the NPC-docked intermediate particles exhibit a central cavity in hexon trimers, the major capsid protein. Quantitative proteomics of isolated nuclei indicated that the intermediate particles bear reduced amounts of fiber and penton-base essential to attach the incoming virus particles to plasma membrane receptors, as well as reduced inner proteins VI and X used for endosomal membrane disruption and charge neutralization of viral DNA, respectively. The intermediate particles colocalized with the host nuclear export factor CRM1/exportin-1, independent of RanGTP hydrolysis required for CRM1 cargo export. They detached from the NPC upon CRM1 inhibition by the small compound leptomycin B, and redocked after inhibitor wash-out, indicating a tunable, direct function of CRM1 in NPC tethering of the intermediate particle poised for DNA uncoating. The results have implications on anti-viral strategies, gene therapy and synthetic biology.", "dataProcessingProtocol" : "Raw files were analyzed using the Fragpipe v19 software. Spectral libraries were generated using MSFragger-DIA, and quantified using DIA-NN (Version 16). Spectra were searched against the UniProtKB Homo sapiens (UniProt ID UP000005640) and AdV-C5 (UniProt ID UP000004992). Carbamidomethylation of cysteines was set as a fixed modification. N-terminal acetylation and oxidation of methionine were searched as dynamic modifications. Differential expression analysis was conducted using the R-package prolfqa. Viral peptides were quantified relative to their hexon and protein VII spectral intensities.", "sampleProcessingProtocol" : "Four million Mib1-KO cells were seeded per 10-cm dish for 24h, inoculated with 73 µg of AdV-C5 in infection medium at 37°C for 120min, washed, and incubated in fresh infection medium for 5h. Nuclei from infected and non-infected cells were NP40-extracted and non-infected ones spiked-in with purified AdV-C5 as described above. Final extracts were re-suspended in 4% SDS, 100 mM Tris 8.1, boiled for 10min, sonicated 3x with a Hielscher sonicator (power 100%, C 60%, amplitude 75%), and subjected to protein concentration determination by Micro BCA assay (Thermo Fisher Scientific). Fifty micrograms of protein were reduced and alkylated by adding Tris-(2-caboxyethyl)phosphine and 2-chroroacetamide to a final concentration of 5 mM and 15 mM, respectively, and incubated at 30°C for 30min. Proteins were diluted with pure ethanol (final concentration 60% v/v EtOH), bound to 500 µg of carboxylated magnetic beads (Cytiva) at RT for 30min, washed 3x with 80% EtOH, and enzymatically digested with 10 µg of trypsin in 50 mM Triethylammonium Bicarbonate (TEAB) buffer at 37°C o/n. Peptides were dried in a SpeedVac vacuum concentrator (Thermo Fisher Scientific), and reconstituted in 3% acetonitrile, 0.1% formic acid. Peptide concentration was determined via Lunatic UV/Vis absorbance spectrometer (Unchained Lab). Two hundred nanograms of peptides were separated on a M-class UPLC and analyzed on an Orbitrap mass spectrometer (Thermo Fisher Scientific) by data-independent acquisition (DIA). ", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-14", "updatedDate" : "2026-04-09", "submissionDate" : "2026-04-09", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Alfonso Gomez Gonzalez" ], "labPIs" : [ "Urs Greber" ], "affiliations" : [ "Department of Molecular Life Sciences University of Zurich Winterthurerstrasse 190 CH-8057 Zurich Switzerland" ], "instruments" : [ "orbitrap" ], "softwares" : [ "FragPipe", "DIA-NN" ], "quantificationMethods" : [ "Relative quantification" ], "sampleAttributes" : [ "cell culture", "Homo sapiens (Human)", "Human adenovirus C serotype 5 (HAdV-5) (Human adenovirus 5)", "epithelial cell" ], "organisms" : [ "Homo sapiens (human)", "Human adenovirus c serotype 5 (hadv-5) (human adenovirus 5)" ], "organismsPart" : [ "Cell culture", "Epithelial cell" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-independent acquisition" ], "sdrf" : "", "projectFileNames" : [ "20230725_C32368_002_S537020_NI_1_Group_1.raw", "20230725_C32368_009_S537030_LMB_3_Group_3.raw", "20230725_C32368_004_S537024_AdV_1_Group_2.raw", "20230725_C32368_015_S537025_AdV_2_Group_2.raw", "20230725_C32368_011_S537026_AdV_3_Group_2.raw", "checksum.txt", "20230725_C32368_016_S537027_AdV_4_Group_2.raw", "20230725_C32368_010_S537029_LMB_2_Group_3.raw", "20230725_C32368_017_S537021_NI_2_Group_1.raw", "20230725_C32368_003_S537023_NI_4_Group_1.raw", "20230725_C32368_014_S537031_LMB_4_Group_3.raw", "diann-output.zip", "20230725_C32368_005_S537028_LMB_1_Group_3.raw", "20230725_C32368_008_S537022_NI_3_Group_1.raw" ], "highlights" : { } }, { "accession" : "PXD076789", "title" : "Phosphoproteomics of tau from tau knockout mice expressing tau in hippocampal neurons during spatial reference learning", "projectDescription" : "This dataset describes the mass spectrometric identification of phosphorylation sites on tau protein isolated from the hippocampus of tau knockout (tau‑/‑) mice during spatial reference learning. Tau expression was selectively reintroduced into hippocampal neurons using adeno‑associated viral (AAV) vectors to enable controlled investigation of tau phosphorylation under defined behavioural conditions. Tau‑/‑ mice received bilateral injections into the dorsal and ventral hippocampus with either AAV‑syn1‑d2tTA‑tau or the control vector AAV‑syn1‑d2tTA‑eGFP. Following postoperative recovery, mice were trained in a spatial reference learning paradigm (day 2 of Morris water maze). Temporal control of tau or eGFP expression was achieved using the doxycycline-regulated d2tTA system, with induction on day 2 of training. Hippocampal tissue was collected at this time point for biochemical analysis. Tau protein was immunoprecipitated from hippocampal lysates and subjected to bottom‑up proteomic analysis. Immunoprecipitated material was reduced, alkylated, and digested with trypsin. Phosphopeptides were subsequently enriched using titanium dioxide (TiO₂) affinity chromatography. Enriched phosphopeptides underwent buffer exchange, vacuum centrifugation, and resuspension prior to liquid chromatography–tandem mass spectrometry (LC‑MS/MS) analysis.", "dataProcessingProtocol" : "Peak lists were generated using MASCOT Distiller and searched with the MASCOT search engine (versions v3.0.0 and v3.1.0; Matrix Science). Searches were performed against the Swiss‑Prot protein sequence database (release 19 January 2024; 570,420 sequences; 206,321,560 residues) restricted to Mammalia taxonomy. Database searches were conducted using the MS/MS ion search type with semi‑tryptic enzyme specificity, allowing up to three missed cleavages. Carbamidomethylation of cysteine residues was specified as a fixed modification, while acetylation of asparagine, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine residues were included as variable modifications. Precursor ion mass tolerance was set to ±5 ppm and fragment ion mass tolerance to ±0.05 Da. The default Mascot minimum peptide length (7 amino acid residues) was applied.", "sampleProcessingProtocol" : "Tau protein was immunoprecipitated from hippocampal tissue extracts adjusted to a total protein concentration of 6 µg/µl. Extracts were incubated with 2 µg of tau‑5 antibody (AHB0042, Invitrogen) for 4 h at 4 °C with gentle rotation. Subsequently, 40 µl of protein A/G agarose beads (20421, Pierce) were added, and samples were incubated for an additional 1 h at 4 °C with rotation. Beads were washed three times with RIPA buffer for 5 min at 4 °C, followed by one rinse and one wash with 20 mM Tris‑HCl (pH 7.4), 2 mM CaCl₂, and 150 mM NaCl. To eliminate residual detergent and protease inhibitors, beads were transferred to a new tube and washed twice with the same Tris‑based buffer, followed by three washes with freshly prepared 20 mM ammonium bicarbonate containing 150 mM NaCl. Beads were then resuspended in 1× trypsin digestion buffer (V5111, Promega). On‑bead proteins were reduced with 1 mM tris(2‑carboxyethyl)phosphine (TCEP) for 10 min at 56 °C and alkylated with 10 mM iodoacetamide (I1149, Sigma‑Aldrich) for 30 min at ambient temperature in the dark. Proteolytic digestion was performed on‑bead using sequencing‑grade trypsin (V5111, Promega) at a 1:25 (w/w) enzyme‑to‑protein ratio for 16 h at 37 °C, supplemented with ProteaseMAX surfactant (V2072, Promega). Phosphopeptides were enriched using TiO₂ spin tips (GL Sciences) according to the manufacturer’s instructions. Trifluoroacetic acid (TFA) was added to the enriched samples to a final concentration of 0.1%. Peptides were desalted using tC18 cartridges (WAT036805, Waters), which were conditioned sequentially with 100% acetonitrile, 50% acetonitrile containing 0.5% acetic acid, and 0.1% TFA. After sample loading, cartridges were washed with 0.1% TFA and 0.5% acetic acid, and phosphopeptides were eluted with 80% acetonitrile containing 0.5% acetic acid. Eluted peptides were dried by vacuum centrifugation (Eppendorf) for 45 min at 45 °C and resuspended in 0.2% heptafluorobutyric acid containing 1% formic acid. Samples were stored at –80 °C until analysis. Phosphopeptides were analysed by LC‑MS/MS using an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) equipped with a FAIMS Pro Duo ion source and coupled to a Vanquish Neo UHPLC system (Thermo Electron). Peptides (3–6 µg injected in 1–7 µl) were first captured on a PepMap Neo C18 trap cartridge (300 µm, 5 µm; Thermo Scientific) and separated on a C18 reverse‑phase analytical column (~25 cm; ReproSil‑Pur, 1.9 µm, 200 Å; Dr. Maisch GmbH) housed in a column oven maintained at 45 °C (Sonation). Peptides were eluted over a 40‑min linear water–acetonitrile gradient containing 0.1% formic acid at a flow rate of 200 nl/min.", "projectTags" : [ ], "keywords" : [ "Spatial reference learning", "Phosphoproteomics", "Tau", "Hippocampus" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-10", "updatedDate" : "2026-04-09", "submissionDate" : "2026-04-09", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Arne Ittner" ], "labPIs" : [ "Arne Ittner" ], "affiliations" : [ "Flinders Health and Medical Research Institute, College of Medicine and Public Health, Flinders University, Adelaide, Australia" ], "instruments" : [ "4800 Plus MALDI TOF/TOF" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "brain", "Mus musculus (Mouse)" ], "organisms" : [ "Homo sapiens (human)", "Mus musculus (mouse)" ], "organismsPart" : [ "Brain" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "F014327.dat", "AP041124_Arne_4.raw", "AP041124_Arne_5.raw", "AP041124_Arne_6.raw", "F014325.dat", "F014329.dat", "AP041124_Arne_3.raw", "F014331.dat" ], "highlights" : { } }, { "accession" : "PXD076809", "title" : "An Integrated Multi-omics Analysis Reveals PDCoV Hijacks the PPAR Signaling Network to Orchestrate Metabolic-Immune Dysregulation and Intestinal Injury in Piglets", "projectDescription" : "Background: Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that causes severe diarrhea and high mortality in neonatal piglets. The molecular mechanisms underlying PDCoV pathogenesis in the host intestine remain poorly understood. Objective: To characterize the host transcriptional, proteomic, and metabolomic responses to PDCoV infection in the primary target organ (jejunum) using a neonatal piglet model.", "dataProcessingProtocol" : "Vendor’s raw MS files were processed using Spectronaut software (19.0.240604.62635) and the built in Pulsar search engine. MS spectra lists were searched against their species level UniProt FASTA databases (uniprotkb_Sus scrofa_9823_2025_03_05.fasta),Carbamidomethyl [C] as a fixed modification, Oxidation (M) and Acetyl (Protein N term) as variable modifications. Trypsin was used as proteases. A maximum of 2 missed cleavage(s) was allowed. The false discovery rate (FDR) was set to 0.01 for both PSM and peptide levels.Peptide identification was performed with an initial precursor mass deviation of up to 20 ppm and a fragment mass deviation of 20 ppm. All the other parameters were reserved as default.", "sampleProcessingProtocol" : "Fifteen-day-old piglets were orally inoculated with PDCoV (USA/Nebraska145/2015 strain) or sterile saline (control). Jejunal tissues were collected at 3 days post-inoculation.For each sample, 200 ng of total peptides were separated and analyzed with a nano UPLC(nanoElute2) coupled to a timsTOF Pro2 instrument (Bruker) with a nano electrospray ion source. Separation was performed using a reversed phase column (PePSep C18, 1.9 μm, 75 μm × 15 cm, Bruker,Germany). Mobile phases were H2O with 0.1% FA (phase A) and ACN with 0.1% FA (phase B). The mass spectrometer adopts DIA PaSEF mode for DIA data acquisition, and the scanning range is from 400 to 1200 m/z, the Ramp time is 100 ms, the Accu time is 100 ms. During PASEF MS/MS scanning, the impact energy increases linearly with ion mobility, from 20 eV(1/K0 = 0.85 Vs/cm2) to 59 eV (1/K0 = 1.30 Vs/cm2).", "projectTags" : [ ], "keywords" : [ "Pdcov", "Jejunum" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-21", "updatedDate" : "2026-04-09", "submissionDate" : "2026-04-09", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Xingchen Guo" ], "labPIs" : [ "guo xing chen" ], "affiliations" : [ "College of Animal Science/Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education, Guizhou University" ], "instruments" : [ "autoflex" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "disease by infectious agent", "jejunum", "epithelial cell", "Sus scrofa domesticus (domestic pig)" ], "organisms" : [ "Sus scrofa domesticus (domestic pig)" ], "organismsPart" : [ "Jejunum", "Epithelial cell" ], "diseases" : [ "Disease by infectious agent" ], "references" : [ ], "experimentTypes" : [ "Data-independent acquisition" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "Jej_10_Slot2-23_1_14640.d.zip", "Jej_4_Slot2-21_1_14638.d.zip", "Jej_3_Slot2-20_1_14637.d.zip", "Jej_12_Slot2-25_1_14642.d.zip", "Jej_11_Slot2-24_1_14641.d.zip", "Jej_2_Slot2-19_1_14636.d.zip", "Jej_5_Slot2-22_1_14639.d.zip", "20250510_100050_651.sne" ], "highlights" : { } }, { "accession" : "PXD076807", "title" : "Detection of KDM1A in cervicovaginal samples from women with high-grade serous ovarian carcinoma: an exploratory pilot study.", "projectDescription" : "Exploratory biomarker studies of high-grade serous ovarian carcinoma (HGSOC) remain challenging in gynaecological oncology due to the lack of reliable, non-invasive diagnostic approaches. Recent evidence suggests that the cervical microenvironment may reflect distal ovarian malignancy through a \"field effect,\" manifesting as epigenetic and proteomic alterations. This study investigates the potential of cervicovaginal sampling (CVS) for HGSOC detection by profiling proteins involved in epigenetic regulation.", "dataProcessingProtocol" : "Protein pellets were denatured in 8 M urea in 0.1 mM TEAB, reduced with 5 mM TCEP at 56°C for 30 min, and alkylated with 10 mM iodoacetamide for 30 min in the dark at 25 °C. Proteins were digested with Lys-C (1:1000, Wako) in 8 M urea for 6 h at 37 °C, followed by dilution to 2 M urea and overnight trypsin digestion (1:100, Promega) at 37 °C. Tryptic peptides were analysed using an Ultimate 3000 nano-RSLC system coupled online to an FAIMS Exploris 480 mass spectrometer (Thermo Scientific). Separation was performed on a C18 nano-column with a 90 min run and a 50 min linear gradient of 5–25% solvent B (A: 0.1% formic acid; B: 0.1% formic acid in 80% ACN). Data were acquired in data-dependent mode with FAIMS voltages of −40 V and −50 V. Spectra were searched against the Homo sapiens UniProt Reference proteome ((20,597sequences) using Proteome Discoverer 2.5 (Thermo Fisher Scientific). Mass tolerances were set to 20 ppm (precursor) and 0.02 Da (fragment). Trypsin specificity allowed up to two missed cleavages. Variable modifications included oxidation (M, +15.995), N-terminal pyro-Glu (Q, -17.027 Da), N-terminal -Met-loss (M, -131.040 Da), -Met-loss + acetyl (M, -89.030 Da), -acetyl (M, +42.011Da); carbamidomethyl (C, 57.021 Da) was set as fixed. Results were filtered at FDR < 1%. Statistical analyses were performed with Perseus [15]. Intensities were log2- transformed and sample-wise median-normalised; missing values were imputed using MNAR settings (width = 0.3, downshift = 1.8) in Perseus to account for low-abundance proteins below the detection limit. Quantification was based on extracted ion chromatograms (XICs) using the Precursor Ions Quantifier node.", "sampleProcessingProtocol" : "Proteins were extracted from the second cell pellet using the Qproteome FFPE Tissue Kit (Qiagen), following the manufacturer’s instructions, which are optimised for formalin-fixed and liquid-based cytology samples. Briefly, the pellets were subjected to thermal denaturation and chemical lysis to release cellular proteins. After centrifugation, the protein-containing supernatant was collected, and protein concentrations were determined using the Bradford assay, with bovine serum albumin as a standard.", "projectTags" : [ ], "keywords" : [ "High-grade serous ovarian carcinoma; kdm1a; cervicovaginal sampling; biomarker; cancer field effect" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-10", "updatedDate" : "2026-04-09", "submissionDate" : "2026-04-09", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Luc Negroni" ], "labPIs" : [ "Frederic DELOM" ], "affiliations" : [ "Université de Bordeaux, Institut Bergonié, Inserm U1312, Bordeaux,France" ], "instruments" : [ "Orbitrap Exploris 480" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "malignant neoplasm of ovary", "endocervical epithelium", "Homo sapiens (Human)" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Endocervical epithelium" ], "diseases" : [ "Malignant neoplasm of ovary" ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "241106_FD_06.mzML", "Samples_description.xlsx", "241106_FD_08.raw", "241106_FD_03.mzML", "241106_FD_all.msf", "241106_FD_07.raw", "241106_FD_05.raw", "241106_FD_03.raw", "checksum.txt", "241106_FD_04.mzML", "241106_FD_07.mzML", "241106_FD_05.mzML", "241106_FD_06.raw", "241106_FD_08.mzML", "241106_FD_02.mzML", "241106_FD_02.raw", "241106_FD_04.raw" ], "highlights" : { } }, { "accession" : "PXD076745", "title" : "Anti-Flag AP-MS of cytoplasmic 3×Flag-importin α1 complexes in HeLa S3 cells", "projectDescription" : "To identify binding partners of importin α1 in the cytoplasm, co-immunoprecipitation using an anti-Flag antibody in the cytoplasmic fraction of 3×Flag–importin α1-inducible HeLaS3 cells.", "dataProcessingProtocol" : "The MS and MS/MS data were searched against the SwissProt database (2021_02) using Proteome Discoverer ver. 2.4 (Thermo Fisher Scientific) with MASCOT search engine software ver. 2.8.0 (Matrix Science). The search parameters were as follows: taxonomy, Homo sapiens; enzyme, trypsin; static modifications, carbamidomethyl (C); dynamic modifications, oxidation (Met) and Acetyl (protein N terminus); precursor mass tolerance, 6 ppm; fragment mass tolerance, 20 mmu; maximum missed cleavage sites, two; and quantitation, label-free quantitation. Proteins were considered to be identified when their false discovery rates were less than 5%. The total amount of each sample was normalized to the total peptide amount using Proteome Discoverer ver. 2.4. Proteins that were five times more abundant in the samples with doxycycline than those without doxycycline were defined as importin α1- specific binding proteins. As a result, 194 proteins were identified. These proteins were ordered according to the abundances of the samples with doxycycline subtracted by those without doxycycline. Prediction of the presence of NLSs was performed using cNLS Mapper (http://nls-mapper.iab. keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) with conditions of a cut-off score of 5.0. The localizations of these identified proteins were categorized according to information from the Human protein Atlas (https://www.proteinatlas.org/).", "sampleProcessingProtocol" : "To prepare the cytoplasmic fraction, HeLa S3/TetOn-3×Flag–importin α1 cells, which can inducibly express 3×Flag–importin α1, were passaged at the density of 8×10^6 cells per 100 mm dish with or without 100 ng/ml doxycycline. After 24 h, the cells were trypsinized, and the cell numbers were counted. Then, 2×10^7 cells were resuspended in 1 ml of TB and transferred into a 2 ml tube. After centrifugation at 2000 g for 30 s, the supernatant was removed. The cells were mixed by gentle pipetting with 1.6 ml of 100 μg/ml digitonin in TB (+PI), which is TB supplemented with a tablet of protease inhibitor (11873580001; Merck) per 10 ml, and incubated at 25°C for 5 min. After centrifugation at 2000 g for 30 s, the supernatant was collected and stored at −30°C. After 20 μl of 50% anti-FLAG M2 affinity gel beads (A2220; Merck) was washed with 1 ml of ice-cold TB (+PI) twice, Anti-FLAG M2 affinity gel was resuspended with 10 μl of ice-cold TB (+PI). Then, it was mixed with the cytoplasmic fraction thawed by block incubator at 25°C, followed by incubation at 37°C with gentle rotation for 2 h. Then, 200 μl ice-cold TB was added and centrifuged at 2000 g for 1 min followed by removal of the supernatants. This washing was repeated three times. The beads were mixed in 100 μl of digestion buffer (50 mM NH4HCO3, 10% CH3CN and 20 mM dithiothreitol) and incubated at 56°C for 30 min. After cooling down to 37°C, the sample was mixed with 10 μl of 333 mM iodoacetamide in 50 mM NH4HCO3 and incubated in the dark at 37°C for 30 min. Then, the sample was mixed with 10 μl of 33 ng/μl trypsin in 50 mM NH4HCO3 and incubated at 37°C overnight. After centrifugation at 2000 g for 30 s, the supernatant was used as a sample for following quantitative mass spectrometry. The experimental pair of samples with and without doxycycline was repeated three times. For liquid chromatography (LC)–tandem mass spectrometry (MS/MS), the trypsinized peptides were subjected to LC (EASY-nLC 1000; Thermo Fisher Scientific) coupled to a Q Exactive quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) with a nanospray ion source in positive mode. LC was performed on a NANO-HPLC capillary column C18 (75 μm×150 mm, 3 μm particle size, Nikkyo Technos) at 45°C. The peptides were eluted with a 100 min 0–30% acetonitrile gradient and a subsequent 20 min 30–65% gradient in the presence of 0.1% formic acid at a flow rate of 300 nl/min. The Q Exactive-MS was operated in the top 10 data dependent scan mode. The parameters for the Q Exactive operation were as follows: spray voltage, 2.3 kV; capillary temperature, 275°C; mass range (m/z), 350–1800; and normalized collision energy, 28%. Raw data were acquired using Xcalibur software.", "projectTags" : [ ], "keywords" : [ "Nls", "Importin", "Nuclear localization signal", "Helas3" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-22", "updatedDate" : "2026-04-08", "submissionDate" : "2026-04-08", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Yutaka Ogawa" ], "labPIs" : [ "Naoko Imamoto" ], "affiliations" : [ "Cellular Dynamics Laboratory, RIKEN Cluster for Pioneering Research (CPR),Saitama 3510198, Japan. Present address:Jikei University of Health Care Sciences, Graduate School of Medical Safety Management, Osaka, Japan." ], "instruments" : [ "Q Exactive" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "endocervical adenocarcinoma", "epithelial cell" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Epithelial cell" ], "diseases" : [ "Endocervical adenocarcinoma" ], "references" : [ "null--pubMed:0--doi: 10.1242/JCS.264517" ], "experimentTypes" : [ "Affinity purification coupled with mass spectrometry proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "211220_QE_240_Ogawa_01-06.pdResult", "211220_QE_240_Ogawa_03-04.pdResult", "211220_QE_240_Ogawa_03_2_negative.raw", "211220_QE_240_Ogawa_05_3_negative.raw", "211220_QE_240_Ogawa_01-02.pdResult", "211220_QE_240_Ogawa_06_3_positive.raw", "211220_QE_240_Ogawa_05-06.pdResult", "211220_QE_240_Ogawa_02_1_positive.raw", "211220_QE_240_Ogawa_01_1_negative_2nd.raw", "211220_QE_240_Ogawa_04_2_positive.raw" ], "highlights" : { } }, { "accession" : "PXD076605", "title" : "Weddell seal milk and serum LC-MSMS", "projectDescription" : "Within any population, some individuals perform better than others. These individuals may survive longer or produce more offspring. In this project we investigate the physiology, behavior, and genetics of female Weddell seals in Erebus Bay, Antarctica. This dataset includes a proteomic comparison of milk and serum in mom-pup pairs across the timecourse of lactation. We also investigate differences between female Weddell seals with a history of frequently producing pups, versus females that have produced pups only infrequently.", "dataProcessingProtocol" : "Data analysis and spectral library generation were performed using Spectronaut (version 16.1.220730, Biognosys AG, Schlieren, Switzerland). A chromatographic library was generated for each of the milk and the serum sample sets from the biological sample DIA runs as well as DIA runs acquired from pooled samples (3 samples from serum for the serum library, 3 samples from milk for the milk library). The species-specific FASTA database for the RefSeq Weddell seal proteome (GCF_000349705.1) was supplemented with a UniProt contaminant database (Crap_uniprot_with_human_MRSonbeadV2). Enzyme specificity was set to trypsin/P, allowing up to two missed cleavages. Variable modifications considered were: Carbamidomethylation C and oxidation M. Peptide precursor and protein identifications were filtered to a false discovery rate (FDR) of 1% (q-value ≤ 0.01). Default Spectronaut settings were used unless otherwise specified. DIA data were analyzed in Spectronaut using the generated spectral library relevant to each tissue. Precursor-level quantification was based on MS2 fragment ion peak areas, as reported in the exported precursor tables (EG-level), which include peptide sequence, precursor identifiers, calibrated precursor m/z, charge state, and associated q-values. An assay-level export containing precursor and fragment ion information (including fragment m/z, ion type, charge, and retention time information) is provided to support reproducibility of DIA signal extraction. Retention time information is reported as observed apex retention time (EG.ApexRT), with normalized iRT values (EG.iRTPredicted) also included. Protein-level quantification was derived from aggregated precursor intensities and is reported as normalized abundance values (PG.Quantity). Missing values were handled using the default imputation strategy implemented in Spectronaut.", "sampleProcessingProtocol" : "Milk samples were centrifuged to separate proteins from lipids prior to protein precipitation. Protein (100 µg per sample) was reduced, alkylated, and digested using a trypsin/Lys-C protease mix (1:10 enzyme-to-protein ratio; Thermo Scientific, #A40006). Peptides were reconstituted in 0.1% formic acid to a final concentration of 1 µg/µL. Liquid chromatography was performed using an UltiMate 3000 RSLCnano system (Thermo Scientific). Peptides were trapped on a C18 PepMap 100 trap column (300 µm i.d. × 5 mm) and separated at 500 nL/min over 60 minutes on a 50 cm uPAC C18 nano-LC column using an EasySpray source and 30 µm ID emitter. Separation was achieved using a 1–45% gradient of solvent B (acetonitrile, 0.1% formic acid) in solvent A (0.1% formic acid). Mass spectrometry was performed on an Eclipse Tribrid Orbitrap instrument using data-independent acquisition (DIA). DIA runs were conducted on each biological sample using a staggered window scheme of 8 m/z over a mass range of 385-1015 m/z. Precursor isolation was performed in the Orbitrap at 60,000 resolution with a dynamic maximum injection, allowing for a minimum of nine points across the peak, a custom automatic gain control (AGC) normalized to 1000% and an NCE of 33 using HCD. Pooled biological samples were also analyzed using DIA in triplicate using the same LC gradient and instrument settings.", "projectTags" : [ ], "keywords" : [ "Reproduction", "Lactation", "Leptonychotes", "Antarctic", "Pinniped" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-10", "updatedDate" : "2026-04-04", "submissionDate" : "2026-04-04", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Allyson Hindle" ], "labPIs" : [ "Allyson Hindle" ], "affiliations" : [ "School of Life Sciences, University of Nevada Las Vegas, USA" ], "instruments" : [ "orbitrap" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "blood serum", "milk", "Phocidae" ], "organisms" : [ "Leptonychotes weddellii", "Phocidae" ], "organismsPart" : [ "Serum", "Blood serum", "Milk" ], "diseases" : [ "Normal" ], "references" : [ ], "experimentTypes" : [ "Data-independent acquisition" ], "sdrf" : "19-nov-22 Early lactation 19101 28756 Male 29024 Oxidation; carbamidomethyl Trypsin/p Pre-weaning Orbitrap eclipse Female Adult Serum Post lactation 27-oct-22 28271 Milk 21-jan-23 High 16-jan-23 18-jan-23 Leptonychotes weddellii 14-dec-22 15253 Data independent acquisition 15330 Hcd Late lactation Normal 1 10-dec-22 2 29055 Label free sample Low 13-nov-22 15-nov-22 11-nov-22 25-nov-22 13-dec-22 7-dec-22 29053 15318 15934", "projectFileNames" : [ "Serum_20230505_22_LW22-30_AF_26.mzML", "Serum_20230505_23_LW22-31_AF_27.raw", "Serum_20230505_19_LW22-18_AF_09.raw", "Serum_20230505_24_LW22-32_AF_17.raw", "Milk_20230531_01_01.raw", "Serum_20230505_06_LW22-05_P_M_16.mzML", "Serum_20230505_21_LW22-26_AF_18.raw", "Milk_20230531_15_08.mzML", "Milk_20230531_10_06.mzML", "checksum.txt", "Serum_20230505_12_LW22-10_P_F_15.raw", "Serum_20230505_14_LW22-14_P_M_02.raw", "Milk_Spectronaut_library.tsv", "Milk_precursor_quant.tsv", "Serum_20230505_13_LW22-14_AF_10.raw", "Milk_20230531_08_05.mzML", "Milk_20230531_10_06.raw", "Serum_precursor_quant.tsv", "Serum_20230505_05_LW22-05_AF_25.mzML", "Serum_20230505_lib03_23.raw", "Serum_20230505_14_LW22-14_P_M_02.mzML", "Serum_20230505_01_LW22-01_AF_24.raw", "Milk_20230531_05_03.mzML", "Serum_20230505_06_LW22-05_P_M_16.raw", "Serum_20230505_04_LW22-04_P_M_13.raw", "Serum_20230505_18_LW22-17_P_F_08.mzML", "Milk_20230531_14_07.raw", "Serum_20230505_02_LW22-01_P_F_06.raw", "Serum_20230505_07_LW22-07_AF_11.raw", "Serum_20230505_24_LW22-32_AF_17.mzML", "Serum_20230505_10_LW22-08_P_F_04.raw", "Milk_lib_20230531_03.raw", "Serum_20230505_20_LW22-18_P_M_19.raw", "Serum_20230505_04_LW22-04_P_M_13.mzML", "Serum_20230505_lib02_12.mzML", "Serum_20230505_15_LW22-15_AF_22.mzML", "Serum_20230505_05_LW22-05_AF_25.raw", "Milk_20230531_08_05.raw", "Serum_20230505_lib02_12.raw", "Serum_20230505_17_LW22-17_AF_20.mzML", "Serum_20230505_21_LW22-26_AF_18.mzML", "Serum_20230505_10_LW22-08_P_F_04.mzML", "Serum_20230505_07_LW22-07_AF_11.mzML", "Serum_20230505_08_LW22-07_P_M_03.raw", "Serum_20230505_16_LW22-15_P_M_21.raw", "Serum_20230505_03_LW22-04_AF_05.mzML", "Serum_20230505_23_LW22-31_AF_27.mzML", "Milk_lib_20230531_02.raw", "Milk_20230531_01_01.mzML", "Milk_20230531_07_04.raw", "metadata_LepWed.sdrf.tsv", "Serum_20230505_15_LW22-15_AF_22.raw", "Serum_20230505_09_LW22_08_AF_07.mzML", "Milk_20230531_17_09.raw", "Serum_20230505_19_LW22-18_AF_09.mzML", "Milk_lib_20230531_01.raw", "Milk_lib_20230531_03.mzML", "Serum_20230505_11_LW22-10_AF_14.mzML", "Serum_20230505_lib01_01.raw", "Milk_20230531_14_07.mzML", "Serum_20230505_09_LW22_08_AF_07.raw", "Serum_20230505_12_LW22-10_P_F_15.mzML", "Serum_20230505_18_LW22-17_P_F_08.raw", "Milk_20230531_05_03.raw", "Milk_protein_groups.tsv", "Serum_20230505_20_LW22-18_P_M_19.mzML", "Serum_20230505_13_LW22-14_AF_10.mzML", "Milk_lib_20230531_02.mzML", "Serum_20230505_17_LW22-17_AF_20.raw", "Serum_Spectronaut_library.tsv", "Serum_20230505_16_LW22-15_P_M_21.mzML", "Serum_20230505_02_LW22-01_P_F_06.mzML", "Milk_20230531_18_10.raw", "Milk_20230531_04_02.mzML", "Milk_20230531_04_02.raw", "Serum_protein_groups.tsv", "Milk_20230531_17_09.mzML", "Serum_20230505_03_LW22-04_AF_05.raw", "Serum_20230505_08_LW22-07_P_M_03.mzML", "Serum_20230505_lib03_23.mzML", "Serum_20230505_lib01_01.mzML", "Milk_20230531_18_10.mzML", "Milk_lib_20230531_01.mzML", "Milk_20230531_15_08.raw", "Serum_20230505_22_LW22-30_AF_26.raw", "Serum_20230505_11_LW22-10_AF_14.raw", "Serum_20230505_01_LW22-01_AF_24.mzML", "Milk_20230531_07_04.mzML" ], "highlights" : { } }, { "accession" : "PXD076596", "title" : "Proteomic analysis of primary human bronchial epithelial cells and extracellular vesicles secreted by polymicrobial culture.", "projectDescription" : "Chronic antibiotic-resistant polymicrobial infections are the leading cause of death in adults with cystic fibrosis (CF). We developed a polymicrobial culture model containing four genera that represents a ‘pulmotype’ detected in ~34% of lung infections in people with CF (pwCF), and accounts for 27% of the variability in lung function. This community, comprised of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica, is grown in synthetic CF media (SCFM2) under anoxic conditions that mimic the environment in mucus plugs in CF. We have shown that Pseudomonas in monoculture communicates with primary human bronchial epithelial cells (pHBEC) by secreting bacterial extracellular vesicles (bEVs) that diffuse through mucus and deliver virulence factors, DNA, and RNA to pHBEC. We report herein that each bacterial genus in the polymicrobial community secretes bEVs containing proteins and RNAs predicted to promote the establishment of chronic infection by enhancing virulence and biofilm formation, and upregulating the stress response and pro-inflammatory pathways in pHBEC. This response is most pronounced in CF pHBEC. Trikafta, a highly effective modulator therapy, does not ameliorate the response or return it to WT levels. Bacterial EVs also inhibited Trikafta-stimulated CFTR Cl- currents by CF pHBEC. These studies provide insight into why Trikafta does not eliminate polymicrobial lung infections and a hyperinflammatory lung environment in pwCF.", "dataProcessingProtocol" : "The raw data files were analyzed by Spectronaut (v. 18.7) using default BGS factory settings in a library-free manner (directDIA+). The human SwissProt database was used for pHBEC samples, and a combined database of P. aeruginosa PA14, P. aeruginosa PA01, S. aureus Newman, S. sanguinis SK36, and P. melaninogenica was used for the polymicrobial bEVs and whole bacteria samples. Peptide spectrum matches, peptides, and protein groups were identified using a false discovery rate (FDR) threshold of 0.01. Resulting protein intensities were analyzed for differential expression with the DEP2 package in R (v4.4.0). Protein expression was filtered and normalized according to default parameters, and missing values imputed with the \"MinDet\" setting. Significant proteins were defined as having an absolute log2 fold change > 1 and an unadjusted P < 0.05 and marked by add_rejections. bEV proteins were analyzed for KEGG pathway activation by ESKAPE Act Plus.", "sampleProcessingProtocol" : "Proteins were isolated from pHBEC treated with PC or bEVs for 6 hrs. Samples were prepared as described previously (PMID: 41696498). Briefly, proteins were isolated in lysis buffer (0.5% SDS, 50 mM AmBic, 50 mM NaCl, HALT Protease Inhibitor) and sonicated. Samples were purified by trichloroacetic acid precipitation. After pelleting and washing with ice‐cold acetone, the resulting protein pellet was re‐suspended in 8 M urea and 0.4 M ammonium bicarbonate, reduced with 4 mM dithiothreitol, and alkylated with 18 mM iodoacetamide. The solution was then diluted to < 2 M urea, and trypsin was added for overnight digestion at 37°C. The resulting peptides were desalted using C18 solid‐phase extraction spin columns, and eluates were dried under vacuum using a SpeedVac concentrator. For mass spectrometry analysis, dried peptides were resuspended in 0.1% formic acid in water (LC-MS grade, Fisher Scientific), and peptides were analyzed using a nanoElute2 coupled with timsTOF Pro2 Mass Spectrometer (Bruker Daltonics). Solvent A was 0.1% FA in water (LC-MS grade, Fisher Scientific), and Solvent B was 0.1% FA in ACN (LC-MS grade, Fisher Scientific). Peptides were separated on a PepSep C18 column (25 cm length, 150 μm I.D., 1.5 μm particle size, 100 Å pore size; Bruker Daltonics), at 600 nL/min flow rate, using a linear gradient of 3% to 25% of Solvent B in 48 min, then raised to 35% B at 54 min, followed by column washing with 95% B. Total method run time was 60 minutes. The source parameters were: capillary voltage = 1700 V, dry gas = 3.0 L/min, and dry temperature = 180 °C. Both the ramp and accumulation times were set to 75 ms. The dia-PASEF method was designed using the py_diAID tool. Briefly, the data was collected in m/z range 300-1200 m/z and mobility range (1/K0) = 0.65-1.35 Vs cm−2 with the following parameters: number of MS1 ramps = 1, number of MS/MS ramps = 14, number of MS/MS windows = 28. The estimated cycle time was 1.20 s.", "projectTags" : [ ], "keywords" : [ "Human bronchial epithelial cells", "Prevotella melaninogenica", "Streptococcus sanguinis strain sk36", "Extracellular vesicles", "Pseudomonas aeruginosa strain pa14", "Pseudomonas aeruginosa strain pa01", "Staphylococcus aureus strain newman" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-13", "updatedDate" : "2026-04-04", "submissionDate" : "2026-04-04", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Samuel Smukowski" ], "labPIs" : [ "Young Ah Goo" ], "affiliations" : [ "Mass Spectrometry Technology Access Center (MTAC), McDonnell Genome Institute, Washington University, St. Louis" ], "instruments" : [ "timsTOF Pro 2" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.64898/2026.04.09.717493" ], "experimentTypes" : [ "Data-independent acquisition" ], "sdrf" : "", "projectFileNames" : [ "26-1265-Barnaby-Stanton-Pro2-Part-2.zip", "checksum.txt", "26-1265-Pro2_Sample01-18.sne", "26-1319-Charpentier-Stanton.sne", "26-1319-Charpentier-Stanton-Pro2.zip", "26-1265_20260106_Sample19_3organism.sne", "26-1265-Barnaby-Stanton-Pro2-Part-1.zip" ], "highlights" : { } }, { "accession" : "PXD076592", "title" : "Proteomic analysis of oleuropein effects in steroid-induced osteonecrosis of osteoblasts", "projectDescription" : "This study investigates the therapeutic effects of oleuropein on steroid-induced osteonecrosis of the femoral head. MC3T3-E1 osteoblasts treated with methylprednisolone were used to establish an in vitro model. TMT-based quantitative proteomics was performed to identify differentially expressed proteins between the model and OLP-treated groups. Functional annotation including Gene Ontology (GO), KEGG pathway, InterPro (IPR), and subcellular localization analyses was conducted. The results indicate that OLP alleviates osteoblast apoptosis and regulates multiple biological processes, particularly the PI3K-AKT signaling pathway.", "dataProcessingProtocol" : "Raw mass spectrometry data were processed using standard proteomics data analysis pipelines. Protein identification and quantification were performed using database search algorithms (e.g., MaxQuant or Proteome Discoverer). The false discovery rate (FDR) was controlled at less than 1% at both peptide and protein levels. Quantitative data were normalized based on TMT reporter ion intensities. Differentially expressed proteins were identified based on fold change and statistical significance criteria. Functional annotation analyses including Gene Ontology (GO), KEGG pathway enrichment, InterPro (IPR) domain annotation, and subcellular localization prediction were performed using publicly available bioinformatics tools.", "sampleProcessingProtocol" : "MC3T3-E1 osteoblasts were cultured and treated with methylprednisolone (MPS) to establish a glucocorticoid-induced injury model, followed by oleuropein (OLP) intervention. Cells were harvested and lysed, and total proteins were extracted. Proteins were reduced, alkylated, and digested with trypsin. The resulting peptides were labeled using Tandem Mass Tag (TMT) reagents. Labeled peptides were fractionated by high-performance liquid chromatography (HPLC) and subsequently analyzed by LC-MS/MS using an Orbitrap mass spectrometer.", "projectTags" : [ ], "keywords" : [ "Proteomics", "Osteoblast", "Osteonecrosis", "Pi3k-akt signaling", "Tmt", "Apoptosis" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-07", "updatedDate" : "2026-04-03", "submissionDate" : "2026-04-03", "downloadCount" : 2, "avgDownloadsPerFile" : 0.25, "botCount" : 1, "hubCount" : 1, "organicCount" : 0, "percentile" : 0, "yearlyDownloads" : [ { "year" : "2026", "count" : 2 } ], "submitters" : [ "LIN lin" ], "labPIs" : [ "Shishui Lin" ], "affiliations" : [ "Fujian Medical University" ], "instruments" : [ "Orbitrap Fusion" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "cell culture", "osteonecrosis", "osteoblast", "Mus musculus (Mouse)" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Cell culture", "Osteoblast" ], "diseases" : [ "Osteonecrosis" ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "1C.txt", "checksum.txt", "2023dbzxl_1.raw", "2023dbzxl_2.raw", "2023dbzxl_3.raw", "EXP.vs.CTL_All.xls", "2C.txt", "3C.txt" ], "highlights" : { } }, { "accession" : "PXD076520", "title" : "qChIP-MS reveals the local chromatin composition by label-free quantitative proteomics", "projectDescription" : "Chromatin immunoprecipitation (ChIP) has been a cornerstone for epigenetic analyses over the last decades, but even coupled to sequencing approaches (ChIP-seq), it is ultimately limited to one protein at a time. In a complementary effort, we here combine ChIP with label-free quantitative mass spectrometry (qChIP-MS) to interrogate local chromatin compositions. We demonstrate the versatility of our approach at telomeres, with transcription factors, in tissue and by dCas9-mediated locus-specific enrichment.", "dataProcessingProtocol" : "Q Exactive HF: Peptides were separated on a C-18-reversed phase column (25 cm length, 75 μm inner diameter; New Objective) which was packed in-house with ReproSil-Pur 120 C18-AQ 1.9 μm resin (Dr Maisch). The column was mounted on an Easy Flex Nano Source and maintained at 40°C by a column oven (Sonation). A 88-min gradient from 2 to 40 % Solution B (80% acetonitrile in 0.1 % (v/v) formic acid) at a flow of 225 nl/min was used, followed by a 12-min washout with 95% Solution B. The spray voltage was set to 2.2 kV. The Q Exactive HF was operated with a TOP20 MS/MS spectra acquisition method per MS full scan, conducted with 60,000 at a maximum injected time of 20 ms and MS/MS scans with 15,000 resolution at a maximum injection time of 75 ms.\n \ntimsTOF fleX: Peptides were separated on an Aurora series column (25 cm length, 75 μm inner diameter, C-18 1.7 μm; IonOpticks) with an integrated captive spray emitter. The column was mounted on a captive spray ionisation source and temperature controlled by a column oven (Sonation) at 50°C. A 88-min gradient from 2 to 40 % (v/v) solution B at a flow of 400 nl/min was used, followed by a 12-min washout with 95% Solution B. The spray voltage was set to 1.65 kV. The timsTOF fleX was operated with data-dependent acquisition (DDA) in PASEF mode with 10 PASEF ramps per topN acquisition cycle (cycle time 1.17s) and a target intensity of 10,000. Singly charged precursor ions were excluded based on their position in the m/z-ion mobility plane and precursor ions that reached the target intensity were dynamically excluded for 24 seconds. \n\nRaw files from both mass spectrometers were processed with MaxQuant  version 2.0.1.0 with preset standard settings for label-free quantitation using at least 2 LFQ ratio counts (except dCas9 ChIP-MS data which was analysed with at least 1 LFQ ratio count) based on the MaxLFQ algorithm . Carbamidomethylation was set as fixed modification while methionine oxidation and protein N-acetylation were considered as variable modifications. Searches were performed against the human (UP000005640) or mouse (UP000000589) Uniprot databases. Search results were filtered with a false discovery rate of 0.01. The resulting MaxQuant proteinGroups tables were further processed with an in-house script (https://github.com/Kappei-Lab/MaxLFQ-Data-Plotting): Known contaminants, proteins groups only identified by site, and reverse hits of the MaxQuant results were removed and only proteins with LFQ intensities were kept. As a default, protein entries with at least four non-zero values in either condition of each pair-wise comparison were included in the analyses. For the ZBTB48 WT/KO ChIP-MS experiments, involving five WT and five KO clones, at least three non-zero values were required. Missing LFQ intensity values were dealt with by randomly imputing from a normal distribution whose parameters were computed from values at or below the lowest 5 % of LFQ intensities in the same set of MaxQuant-processed data, and the average value of three iterations were used. All LFQ volcano plots are based on 4- 5 biological replicates, with x-axis and y-axis of each plot representing the ratio of mean MS2-based intensities between both experimental conditions in log2 scale and p-value in log10 scale, respectively. Every protein group quantified and presented in supplementary tables is represented as a circular dot in their corresponding volcano plot. Number of proteins identified by mass spectrometers and MaxQuant software, along with number of proteins quantified by our in-house analysis and imputation pipeline described above, is indicated within each plot. Number of significant protein hits above the two-dimensional cut-off of >4-fold enrichment and p<0.05 was indicated in the top right corner of each plot. Colour legends are included in respective figure legends.", "sampleProcessingProtocol" : "ChIP-mass spectrometry (ChIP-MS). For each replicate approximately 1.25-5 × 10^7 cells, were mixed with 990-3960 μl PBB1, 2.5-10 μg antibodies and 37.5-150 μl Dynabead protein G in a 5 ml DNA LoBind tube (Eppendorf). For dCas9-GFP ChIP-MS, each reaction consisted of 600 μg DNA sonicate, approximately 10 × 107 cells, 7920 μl PBB1 and 96 μl GFP-trap beads prepared in two 5 ml DNA LoBind tubes (Eppendorf). Protein samples were eluted by resuspending magnetic beads in 25 μl 2× Tris-Glycine Sample Buffer after the final three washes with PBB2. To reverse DSP crosslinks, 2 μl 1 M DTT were added, and samples were incubated at 37°C for 30 min, followed by 10 min at 95°C to reverse FA crosslinks. Denatured samples were stored in -80°C until sample preparation for proteomics analysis.\n\nCasID-MS (BirA* and APEX2). Cells were seeded at a density of 4 x 10^6 in 15 cm dishes for 48 h with 1 µg/mL doxycycline for induction of BirA*-dCas9 or APEX2-dCas9. For BirA*, biotinylation with 50 µM biotin was performed 24 h prior to the end of 48 h induction. For APEX2 labelling, cells were pre-treated with 500 µM biotin-phenol (AdipoGen Life Sciences) for 30 min at 37°C followed by 5 min labelling with 1 mM H2O2. Cells were then washed thrice with quenching solution (5 mM Trolox; Sigma-Aldrich), 10 mM sodium ascorbate (Sigma-Aldrich), 10 mM sodium azide (Sigma-Aldrich) and once with cold PBS to stop the biotinylation reaction. The cells were washed thrice with quenching solution and once with cold PBS. After biotinylation, the nuclear protein extracts of these cells were prepared as mentioned above and the biotinylated proteins were captured using MyOne Streptavidin C1 Dynabeads (Thermo Scientific). In brief, 150 µL of beads were used for each replicate. The beads were first washed and resuspended with PBB+ buffer (420 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 0.25 % (w/v) IGEPAL CA-630, 1 mM DTT, and 1× cOmplete protease inhibitor (Roche)). Next, 250 µL of 4 µg/mL nuclear protein extracts were added to the beads and mixed for 2 h on a rotating wheel at 4 °C. The beads were pelleted and washed thrice with increasing volumes of cold PBB+ buffer before eluting the isolated biotinylated proteins with 25 µL of sample buffer, Laemmli 2× concentrate (Sigma Aldrich, S3401) and boiling at 95 °C for 5 min. Denatured samples were stored in -80°C until sample preparation for proteomics analysis.\n\nLabel-free quantitative proteomics. Denatured ChIP-MS and CasID-MS elutes were separated by SDS-PAGE gel electrophoresis using NuPAGE 12 % Bis-Tris Mini Protein gels (Thermo Scientific) at 160 V for 20-30 min. Each ChIP-MS sample was prepared as two or four fractions for in-gel trypsin digestion. Briefly, gel fractions were cut approximately into 2 by 2 by 1 mm pieces, destained twice using Destaining Solution (50 % (v/v) ethanol, 25 mM ammonium bicarbonate) and dehydrated with acetonitrile. Gel pieces were dried using a speed vacuum concentrator (Eppendorf) and subsequently treated with Reducing Solution (50 mM DTT, 25 mM ammonium bicarbonate) at 56 °C for 1 h followed by Alkylating Solution (55 mM iodoacetamide, 25 mM ammonium bicarbonate) at room temperature for 45 min in the dark. Gel pieces were washed once with 50 mM ammonium bicarbonate and twice with acetonitrile, dried using a speed vacuum concentrator, and incubated with sequencing grade trypsin (Promega) in 50 mM ammonium bicarbonate at 37 °C overnight. Digested peptides were extracted from the gel pieces on the following day through two incubations each using an alternating cycle of Extraction Buffer (3 % (v/v) trifluoroacetic acid, 30 % (v/v) acetonitrile, 25 mM ammonium bicarbonate) and acetonitrile for 20 min and 10 min, respectively, followed by a final incubation in acetonitrile for 10 min. All supernatants were collected and combined. Extracted peptides in solution were concentrated using a speed vacuum concentrator for 2 h, and desalted with home-made stage-tips containing C-18 resins (Empore). Stage tips were stored at 4°C prior elution of peptides for analysis using an EASY-nLC 1200 Liquid Chromatograph (Thermo Scientific) coupled to either a Q Exactive HF (Thermo Scientific) or timsTOF fleX (Bruker) mass spectrometer.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-03", "updatedDate" : "2026-04-02", "submissionDate" : "2026-04-02", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Dennis Kappei" ], "labPIs" : [ "Dennis Kappei" ], "affiliations" : [ "Cancer Science Institute of Singapore (CSI), National University of Singapore (NUS)" ], "instruments" : [ "Q Exactive HF", "timsTOF fleX" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "osteosarcoma" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ ], "diseases" : [ "Osteosarcoma" ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition" ], 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followed by liquid chromatography-tandem mass spectrometry showing EGFR is a potential binding protein of PAI-1", "projectDescription" : "To discover potential binding protein of PAI-1 in human PDAC cells.", "dataProcessingProtocol" : "A Pierce Silver Stain Kit was used to visualize the protein bands. LC-MS/MS was used to analyze bands that displayed notable variations.", "sampleProcessingProtocol" : "A Flag Immunoprecipitation Kit was used for immunoprecipitation. Following cell lysis and centrifugation, the protein lysates were incubated with an anti-Flag affinity gel. Afterwards, the resin was washed using 1×wash buffer and Flag-peptides were added to elute the Flag fusion protein by incubation at 4°C for 3 hours. Finally, the eluents were denatured and subjected to precast gel electrophoresis for immunoblotting analysis.", "projectTags" : [ ], "keywords" : [ "Human", "Pdac", "Panc-1 cell", "Lc-ms/ms" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-02", "updatedDate" : "2026-04-02", "submissionDate" : "2026-04-02", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Jianwei Zheng" ], "labPIs" : [ "Xuan Zhou" ], "affiliations" : [ "Tianjin Medical University Cancer Institute and Hospital" ], "instruments" : [ "Q Exactive" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "cell culture", "Homo sapiens (Human)", "epithelial cell", "pancreatic ductal adenocarcinoma" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Cell culture", "Epithelial cell" ], "diseases" : [ "Pancreatic ductal adenocarcinoma" ], "references" : [ ], "experimentTypes" : [ "Gel-based experiment" ], "sdrf" : "", "projectFileNames" : [ "MM-liuchao-SERPINE1-2-20211208.msf", "MM-liuchao-SERPINE1-3-20211208.msf", "MM-liuchao-SERPINE1-1-20211208.msf", "MM-liuchao-SERPINE1-5-20211208.msf", "MM-liuchao-SERPINE1-4-20211208.msf", "MM-liuchao-SERPINE1-2-20211208.raw", "MM-liuchao-SERPINE1-8-20211208.msf", "MM-liuchao-SERPINE1-6-20211208.msf", "MM-liuchao-SERPINE1-1-20211208.raw", "MM-liuchao-SERPINE1-7-20211208.msf", "MM-liuchao-SERPINE1-8-20211208.raw", "checksum.txt", "MM-liuchao-SERPINE1-6-20211208.raw", "MM-liuchao-SERPINE1-7-20211208.raw", "MM-liuchao-SERPINE1-3-20211208.raw", "MM-liuchao-SERPINE1-5-20211208.raw", "MM-liuchao-SERPINE1-4-20211208.raw" ], "highlights" : { } }, { "accession" : "PXD076477", "title" : "ProjDIA: Fragment-Bin Projection Scoring and Calibrated Confidence for Direct Infusion DIA Proteomics", "projectDescription" : "Direct-infusion shotgun proteome analysis (DI-SPA) increases throughput by removing liquid chromatography, but the resulting DIA spectra are highly multiplexed and difficult to interpret. This dataset was generated to evaluate ProjDIA, a computational workflow for direct-infusion DIA proteomics. ProjDIA uses fragment-bin projection scoring, confidence-guided two-pass mass-error recalibration, monotone q-value reporting, and semi-supervised rescoring to improve peptide and protein identification confidence. The dataset includes benchmarking experiments across MS2 resolution and sample load, replicate-series analyses, species-entrapment controls for external error assessment, and a three-species mixture for label-free quantification evaluation. The submission contains the raw mass spectrometry files together with processed search and result files used to support the analyses reported in the study.", "dataProcessingProtocol" : "Raw files were converted to mzXML and analyzed with ProjDIA using a spectral-library-based workflow. Scans were grouped by isolation-window center, window width, and FAIMS compensation voltage. Candidate matches were generated by fragment matching within a user-defined ppm tolerance, followed by fragment-bin projection scoring. ProjDIA then performed a first-pass target-decoy analysis, confidence-guided mass-error recalibration using high-confidence target matches, a second-pass search with corrected tolerance, and semi-supervised rescoring using multiple match-quality features. Final peptide- and protein-level results were reported using monotone q-values, and protein inference used an IDPicker-style parsimony approach. Quantification analyses used summed matched-fragment intensities with reproducibility filtering based on coefficient of variation and replicate presence. Comparator analyses were performed with CsoDIAq and CHIMERYS using the same benchmark datasets where applicable.", "sampleProcessingProtocol" : "HeLa Standard digest was prepared under water in 0.1% formic acid. The digested peptides were analyzed on Orbitrap Exploris 480 using a direct infusion method operated in data-independent acquisition (DIA) mode. Samples were ionized via cVSSI49–51 or ESI as the ion source, and ions were introduced into the mass analyzer through a heated ion transfer capillary maintained at 320 °C. The DIA method was employed using a resolution setting of 120,000 for MS2 to ensure high mass accuracy and under positive ion mode. The AGC target was set to 300%, with maximum injection time set to Auto to optimize ion accumulation while maintaining an efficient acquisition duty cycle. DIA was performed using a narrow window width of 1 m/z spanning the 380–1000 m/z range. A list of expected precursor m/z values was generated automatically from Xcalibur software. The total acquisition time was approximately 3 minutes. Finally, the .raw file was converted and analyzed using different methods to generate the entrapment evaluation.", "projectTags" : [ ], "keywords" : [ "Hela", "Mcf7", "Direct infusion", "Proteomics", "Projdia", "Di-spa", "Species entrapment", "Dia", "Label-free quantification" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-02", "updatedDate" : "2026-04-02", "submissionDate" : "2026-04-02", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Herbi Yuliantoro" ], "labPIs" : [ "Peng Li" ], "affiliations" : [ "Lehigh University" ], "instruments" : [ "orbitrap" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "epithelial cell", "cervix carcinoma" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Epithelial cell" ], "diseases" : [ "Cervix carcinoma" ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "data19cvssi.psm.q0.01.tsv", "data12cvssi.protein.q0.01.tsv", "data18cvssi.peptide.q0.01.tsv", "data10cvssi.raw", "data19cvssi.raw", "data15ESI.peptide.q0.01.tsv", "data14cvssi.mzXML", "checksum.txt", "data9cvssi.raw", "data10cvssi.peptide.q0.01.tsv", "data14cvssi.peptide.q0.01.tsv", "data16ESI.peptide.q0.01.tsv", "data11cvssi.protein.q0.01.tsv", "data14cvssi.raw", "data10cvssi.psm.q0.01.tsv", "data13cvssi.psm.q0.01.tsv", "data9cvssi.peptide.q0.01.tsv", "data11cvssi.psm.q0.01.tsv", "data12cvssi.psm.q0.01.tsv", "data13cvssi.peptide.q0.01.tsv", "data18cvssi.raw", "data19cvssi.protein.q0.01.tsv", "data16ESI.raw", "data9cvssi.psm.q0.01.tsv", "data13cvssi.raw", "target_human_trap_yeast_ecoli.fasta", "data18cvssi.psm.q0.01.tsv", "data14cvssi.protein.q0.01.tsv", "data13cvssi.mzXML", "ecolihumanyeast_concat_mayu_IRR_cons_openswath_64var_curated_commas_with_decoys.tsv", "data10cvssi.mzXML", "data17ESI.mzXML", "data10cvssi.protein.q0.01.tsv", "data16ESI.psm.q0.01.tsv", "data12cvssi.raw", "data14cvssi.psm.q0.01.tsv", "data18cvssi.protein.q0.01.tsv", "data15ESI.protein.q0.01.tsv", "data18cvssi.mzXML", "data12cvssi.peptide.q0.01.tsv", "data13cvssi.protein.q0.01.tsv", "data19cvssi.peptide.q0.01.tsv", "data9cvssi.mzXML", "data11cvssi.mzXML", "data11cvssi.raw", "data16ESI.mzXML", "data19cvssi.mzXML", "data15ESI.raw", "data15ESI.mzXML", "data12cvssi.mzXML", "data16ESI.protein.q0.01.tsv", "data9cvssi.protein.q0.01.tsv", "data15ESI.psm.q0.01.tsv", "human.faims.fixed.download.sample.130941.RefEntrapment.psps23.RefDecoy.psps13.mgf", "data17ESI.raw", "data11cvssi.peptide.q0.01.tsv" ], "highlights" : { } }, { "accession" : "PXD076496", "title" : "Statistical crystallography reveals an allosteric network in SARS-CoV-2 Mpro", "projectDescription" : "To interpret and transmit biological signals, proteins use correlated motions. Experimental determination of these dynamics and the structural distributions they generate remains a key challenge. Here, using 1146 crystal structures of the main protease (Mpro) from SARS-CoV-2, we were able to infer a model of the enzyme’s structural fluctuations. Mpro is regulated by concentration, becoming enzymatically active after forming a homodimer. To understand the structural changes that enable dimerization to activate catalysis, we employed our model, predicting which regions of the dimerization domain are structurally correlated with the active site. Mutations at these positions, expected to disrupt catalysis, resulted in a dramatic reduction in activity in one case, a mild effect in the second, and none in the third. Additional crystallography and biophysical experiments provide a mechanistic explanation for these results. Our work suggests that a statistical crystallography, in which numerous crystallographic datasets are analyzed, can reveal the structural fluctuations of protein native states and help uncover their biological function.", "dataProcessingProtocol" : "Native Mass Spectroemtry. Peaks were assigned either manually or with UniDec and then listed into a peak list. This peak list was used to extract the area under the curve of each peak. These were then summed, normalized and plotted.", "sampleProcessingProtocol" : "Native Mass spectrometry. Using Micro Bio-Spin P-6 columns (Bio-Rad, 6000 MWCO) and performing two cycles, purified wild-type and mutant Mpro was buffer exchanged into 300 mM ammonium acetate pH 8.0 and 1 mM DTT, which preserves non-covalent interactions and known to be compatible with Mpro 6363. All variants were measured at 2 µM. In addition, wild type was measured at 10 µM and N214A at 10 µM and 20 µM.", "projectTags" : [ ], "keywords" : [ "Sars-cov-2", "Enzymology", "Crystallography" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-14", "updatedDate" : "2026-04-02", "submissionDate" : "2026-04-02", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Kira Schamoni-Kast" ], "labPIs" : [ "Charlotte Uetrecht" ], "affiliations" : [ "Centre for Structural Systems Biology CSSB, Deutsches Elektronen-Synchrotron DESY & Leibniz Institute of Virology (LIV) & University of Lübeck, Notkestr. 85, 22607 Hamburg, Germany Institute of Chemistry and Metabolomics, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany" ], "instruments" : [ "Q Exactive" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Escherichia coli" ], "organisms" : [ "Escherichia coli" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Top-down proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "Mpro_Kd_2uM_raw_data.zip", "Mpro_Kd_scans_all_data.zip", "Mpro_mutant_peak_list.xlsx", "Kd_conc_dependence_WT_N214A_scans.zip", "Mpro_Kd_10_20uM_raw_data.zip" ], "highlights" : { } }, { "accession" : "PAD000036", "title" : "Multicohort analysis unveils axon guidance pathways linking small for gestational age to spirometric restriction.", "projectDescription" : "Children born small for gestational age (SGA) face elevated risks of metabolic, cardiovascular, respiratory, and neurodevelopmental disorders, as well as premature mortality, yet the underlying mechanisms remain only partly understood. We analyze blood proteomic data from multiple birth cohorts to identify molecular pathways linked to SGA and to later-life lung function. We find that approximately one-third of SGA children exhibit a distinct molecular endotype marked by dysregulation of axon-guidance proteins in cord blood. In peripheral blood collected later in life, these proteins are inversely associated with contemporaneous spirometric restriction. These findings offer new insight into the developmental origins of chronic disease and highlight axon-guidance pathways as promising targets for investigating multiorgan morbidity. This repository accompanies the publication entitled “Multicohort analysis unveils axon guidance pathways linking small for gestational age to spirometric restriction.”", "dataProcessingProtocol" : "All subsequent data QC and analysis was conducted in the R (version 4.3.2) environment unless otherwise stated. The SomaScan data .adat files for cord and later life sample were imported into R and assessed with the same QC pipeline. Sample RFU and log10(RFU) distribution were visualized with boxplots to detect any aberrant samples. Aptamers other than those corresponding to human proteins were excluded, which were those annotated Hybridization Control Elution, Non-Biotin, Non-Cleavable, Non-Human, Spurimer, and Spuriomer. Aptamer expression was assessed with respect to buffer samples and removed if they had an average expression difference of less than 100 RFU and/or a ratio less than 1.5. This step ensured a gap between aptamer expression and baseline buffer expression and ultimately excluded few aptamers. Outlier samples were assessed with a preliminary Principal Component Analysis (PCA) to visualize separation from the buffer, calibrator, and pooled QC samples. The QC and calibrator samples were assessed to confirm even expression across the sequence order, thus confirming appropriate normalization steps were effective. Buffer, calibrator, and QC samples were then excluded from further quality control. As mentioned above, multiple aptamers can correspond to the same target protein. To assess the overlap of these aptamers, PCA was run on all aptamers and the first two PC were plotted with linkage between aptamers that encode the same target protein. This strategy showed that linked aptamers do not occupy the same principal component space, indicating they represent different aspects (e.g., conformations, subtypes) of the same target protein. Thus, all aptamers with the same protein were retained for analysis (rather than averaged or summed), and analysis was conducted at the aptamer level. For this reason, some analyses show a target protein multiple times, as expected from closely related aptamers corresponding to the same target protein. Aptamer density distributions were also assessed for outlier aptamer expression. Density distribution curves, Relative Log Expression plots, and Cumulative Distribution Function plots were generated for each sample, stratified by relevant technical and biological variables (e.g., assay plate, sex), to assess outlier samples. Additionally, PCA was used again to detect outliers (without buffer, calibrator, and QC samples), and the top 10 PCs were to assess to determine whether they capture variation attributable to any known variables (e.g., Sex). An example QC report is available on the Dryad repository. Following pre-processing and QC, there were 207 Cord blood proteomic profiles with 7,214 aptamers corresponding to 6,536 unique proteins and 139 later life (pediatric/adult) samples with 7,154 aptamers corresponding to 6,480 unique proteins.", "sampleProcessingProtocol" : "The samples used in this study were curated from cohorts in the recently established Children’s Allergy and Asthma Data Repository (CADRE), previously referred to as The Children's Respiratory and Environmental Workgroup (CREW) consortium; a resource to pool and harmonize data from 13 allergy and asthma birth cohort studies in the US. Six of these cohorts are utilized in the present study. Proteomic data was generated with the SomaScan platform (SomaLogic, Boulder, CO, USA), which allows high-throughput, simultaneous quantification of thousands of proteins. The fluorescence-based detection of aptamer abundance is reported as Relative Fluorescent Units (RFU), which is proportional to the concentration in the sample. The term ‘aptamer’ refers to the short, single-stranded oligonucleotides that bind to target proteins with high affinity. Before data generation, the sample order was randomized and checked for even distribution with respect to SGA status and sex to ensure there was no inadvertent grouping by SGA/sex in the sequence order. The data received the following standardization/normalization as part of the data generation process: 1) Hybridization control normalization (to mitigate variation within the run that comes from the readout steps), 2) Median signal normalization (pooled calibrator replicates within run to mitigate technical variation in the calibrator signal), 3) Plate scale (adjusts for overall signal intensity differences between runs), 4) Calibration scale (adjusts for aptamer reagent-specific assay differences between runs), and 5) Median normalization (final sample normalization). The cord and adult data from the TCRS cohort were generated first, with expansion to include cord blood data from the CCCEH, COAST, IIS, and WISC cohorts and childhood data from the COAST, IIS, WHEALS, and WISC cohorts. The childhood data generated from the COAST and WISC cohorts were ultimately excluded from analysis due to missing spirometry data (COAST) and small sample size, (WISC, n = 3). The data was generated using the same methodologies, platform, and chemistry, ultimately profiling the same set of aptamers.", "projectTags" : [ ], "keywords" : [ "Cord blood", "Proteomics", "Small for gestational age", "Somascan" ], "doi" : "10.6019/PAD000036", "submissionType" : "AFFINITY", "publicationDate" : "2026-04-02", "updatedDate" : "2026-04-01", "submissionDate" : "2026-04-01", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "James Read" ], "labPIs" : [ "Anthony Bosco" ], "affiliations" : [ "Associate Professor, Immunobiology Associate Research Scientist, A2DRC The University of Arizona, USA" ], "instruments" : [ "SomaScan assay v4.1" ], "softwares" : [ ], "quantificationMethods" : [ "Relative quantification" ], "sampleAttributes" : [ "Homo sapiens (Human)", "blood" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Blood" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "SomaScan affinity proteomics", "Affinity proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "SS-2217258_v4.1_TCRS_Adult_Serum.hybNorm.medNormInt.plateScale.calibrate.anmlQC.qcCheck.medNormRefSMP.adat", "SS-2342591_v4.1_CADRE_Cord__HeparinPlasma.hybNorm.medNormInt.plateScale.calibrate.anmlQC.qcCheck.medNormSMP.adat", "CADRE_Cord_data_comb.csv", "CADRE_LL_pheno_comb_rm.csv", "CADRE_LL_data_comb_rm.csv", "CADRE_Cord_aptamer_info_comb.csv", "SS-2217260_v4.1_TCRS_Cord_Serum.hybNorm.medNormInt.plateScale.calibrate.anmlQC.qcCheck.medNormRefSMP.adat", "CADRE_LL_aptamer_info_comb.csv", "CADRE_Cord_pheno_comb.csv", "SS-2342592_v4.1_CADRE_Pediatric_HeparinPlasma.hybNorm.medNormInt.plateScale.calibrate.anmlQC.qcCheck.medNormSMP.adat" ], "highlights" : { } }, { "accession" : "PXD076443", "title" : "The H3K4 methyltransferase KMT2D is an essential cofactor for GATA1 at erythroid gene enhancers", "projectDescription" : "Gene expression during cellular differentiation is coordinated by combinatorial interactions between transcription factors (TFs) and cofactors at promoters and enhancers. The “master TF” GATA1 coordinates gene transcription in a subset of hematopoietic lineages, including erythroid, megakaryocytic, mast, and eosinophil, while repressing the development of other blood lineages. However, the specific cofactors required for GATA1-activated gene expression during hematopoiesis are incompletely defined. We identified the cofactor KMT2D, an H3K4 methyltransferase that collaborates with H3K27 acetyltransferases to activate transcription, in an unbiased CRISPR/Cas9 screen for epigenetic regulators of erythropoiesis. Loss of KMT2D in human erythroid precursors caused developmental arrest with impaired expression of numerous erythroid genes. Mechanistically, KMT2D colocalized with GATA1 on more than one thousand erythroid enhancers associated with over two hundred erythroid genes. In general, co-occupancy of GATA1 and KMT2D at erythroid enhancers was associated with stronger transcriptional activity than occupancy by GATA1 alone. Acute depletion of KMT2D in erythroid precursors caused rapid reductions of H3K4me1 and H3K27ac on a subset of GATA1-bound enhancers and impaired the expression of canonical erythroid genes, including ZFPM1, SLC4A1, and EPOR. Moreover, acute depletion of GATA1 or KMT2D individually caused downregulation of overlapping gene sets. Thus, KMT2D controls erythropoiesis by selectively activating GATA1-dependent erythroid enhancers. Our studies identify KMT2D as a novel cofactor for transcriptional activation by GATA1 during erythropoiesis. More generally, our findings demonstrate how a lineage-specific TF cooperates with a ubiquitous epigenetic regulator to drive lineage-specific gene expression during cellular differentiation.", "dataProcessingProtocol" : "The resulting MS/MS data were processed using MaxQuant search engine (v.1.6.15.0). Tandem mass spectra were searched against the Homo_sapiens_9606_SP_20231220.fasta (20429 entries) concatenated with reverse decoy and contaminants database. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. Min. peptide length was set as 7 and max. number of modification per peptide was set as 5. The mass tolerance for precursor ions was set as 20 ppm in first search and 20 ppm in main search, and the mass tolerance for fragment ions was set as 20 ppm. Carbamidomethyl on Cys was specified as fixed modification, and acetylation on protein N-terminal and oxidation on Met were specified as variable modifications. False discovery rate (FDR) of protein, peptide and PSM was adjusted to < 1%.", "sampleProcessingProtocol" : "For co-immunoprecipitation, collect 20M cells from each group of samples and use the nuclear and cytoplasmic extraction kit (Abbkine, #KTP3001) to collect nuclear extracts. Briefly, cells were harvested and then centrifuged at 500 g for 5 min. After washing with ice-cold 1× PBS, 200 L of ice-cold hypotonic buffer [10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 1 mM EDTA, pH 7.9] containing 1× protease inhibitor cocktail (Sigma-Aldrich, P8849) was added to burst the cell pellet, and incubation was carried out on ice for 10 min. Then, 11 L of ice-cold detergent (0.05% NP-40) was added, and the incubation was prolonged on ice for an additional 1 min. After centrifugation (5 min at 16,000 g), the supernatant (cytoplasmic extract) was transferred to a pre-chilled tube while the pellet fraction containing the nuclei was suspended in 100 L of ice-cold nuclear extraction buffer (5 mM HEPES, 1.5 mM MgCl2, 300 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol, pH 7.9) containing 1× protease inhibitor cocktail. The samples were placed on ice and vortexed for 15 s every 10 min for a total of 40 min. After centrifugation (10 min at 16, 000 g), the supernatant (nuclear extract) was collected into a pre-chilled tube. The supernatant was incubated with the indicated antibodies at 4 °C overnight, followed by the addition of protein A/G-coated agarose beads (MCE). Incubate at 4 ℃ for 12 hours and wash three times with PBS for mass spectrometry analysis.", "projectTags" : [ ], "keywords" : [ "Kmt2d; gata1; enhancer; transcription regulation; hematopoiesis; erythropoiesis; epigenetics" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-10", "updatedDate" : "2026-04-01", "submissionDate" : "2026-04-01", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Ye Xin" ], "labPIs" : [ "Ye Xin" ], "affiliations" : [ "Soochow university, China" ], "instruments" : [ "LTQ Orbitrap" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "acute leukemia", "cell suspension culture", "erythrocyte" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Erythrocyte", "Cell suspension culture" ], "diseases" : [ "Acute leukemia" ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "XB04040LI_IG2.raw", "XB04040LI_IG1.raw", "XB04040LI_K2D2.raw", "XB04040LI_K2D1.raw", "evidence.zip" ], "highlights" : { } }, { "accession" : "PXD076459", "title" : "Multiple disulfide-bonded states confer conformational diversity in fibrinogen", "projectDescription" : "Disulfide bonds constrain the polypeptide backbone and reduce conformational variability in proteins. The blood clotting protein fibrinogen is constitutively produced as multiple partially disulfide-bonded states, suggesting that individual fibrinogen molecules have a variety of conformational forms. This hypothesis was tested by resolving fibrinogen molecules on beads coated with different fibrinogen ligands and measuring their disulfide states by differential cysteine alkylation and mass spectrometry. Polyclonal anti-fibrinogen antibodies resolved states where all 11 measured disulfides across the molecule were significantly less formed. In contrast, the GHRP peptide, which binds the fibrinogen β-nodule, resolved states in which seven β-nodule and central E region disulfides were significantly more formed, while the GPRP peptide, which binds the γ-nodule, resolved states in which only one disulfide was significantly more formed. To probe the link between disulfide state and conformation, in silico analysis of all 32 possible disulfide-bonded states of the β-nodule revealed that the conformational flexibility of this domain and predicted GHRP interactions in its binding pocket are predicated on the oxidation state of one of the five β-nodule disulfides, βC424-βC437. These findings indicate that the different disulfide-bonded states of fibrinogen adopt an extensive array of conformations that are selectively recognized by different fibrinogen ligands.", "dataProcessingProtocol" : "MS/MS spectra were searched against the Swissprot reference proteome using Mascot search engine (Version 3.1, Matrix Science). Precursor mass tolerance and fragment tolerance were set at 10 ppm and the precursor ion charge state to 2+ and 3+. Variable modifications were defined as oxidized Met, deamidated Asn/Gln, N-terminal pyro Glu/Gln, Iodoacetanilide derivative cysteine and Iodoacetanilide-13C derivative cysteine with full trypsin cleavage and up to one missed cleavage. Only peptides with a peptide score ≥ 30 (p < 0.05) and error ≤6 ppm were selected for quantification of relative abundance. Ion chromatograms of peptides labeled with 12C-IPA and/or 13C-IPA were generated using FreeStyle software (Thermo Fisher). The area under the curve (AUC) of each peptide was calculated using the automated peak detection function built into Freestyle. The fraction of unformed disulfide bond was calculated as the AUC of 12C-IPA labeled peptide divided by the AUC of 12C-IPA plus 13C-IPA labeled peptides. The data was routinely searched for peptides containing unlabeled cysteine thiols and these were not detected, which indicates that alkylation of unpaired cysteine residues by 12C-IPA or 13C-IPA was complete in the protein.", "sampleProcessingProtocol" : "For some samples, dynabeads were coated with 16 μg of polyclonal anti-fibrinogen antibody in 1 mL of phosphate-buffered saline (PBS) on a rotating wheel for 1 h at 22°C and the excess antibody removed by washing three times with 1 mL of PBS. Plasma samples were incubated with 2 mg of coated beads on a rotating wheel for 1 h at 22°C. The beads were collected using a magnet and incubated in 0.3 mL of 4 mM 2-Iodo-N-phenylacetamide (12C-IPA) in PBS for 1 h at room temperature with gentle rotation, while kept away from light. Control samples of 5 μL plasma were also incubated with 4 mM 12C-IPA in the dark. For some samples, thirty μM of GHRPLC or GPRPLC peptides was incubated with 20 μL of maleimide agarose in 0.5 mL of PBS with 1 mM ethylenediaminetetraacetic acid (EDTA) for 1 h with rotation on a wheel. Agarose was pelleted by centrifugation at 500g and washed with 0.5 mL of PBS three times prior to blocking for 1 h with 50 mM glutathione in PBS with 1 mM EDTA and three more washes with 0.5 mL of PBS. Control (no peptide) or peptide-labelled agarose was centrifuged, resuspended in 500 μL of plasma and incubated for 1 h with rotation at room temperature. The agarose was centrifuged, the beads washed three times in 0.5 mL of PBS and resuspended in 0.5 mL of 4 mM 2-Iodo-N-phenylacetamide (12C-IPA) in PBS for 1 h at room temperature with gentle rotation in the dark. Control samples of 5 μL plasma were also incubated with 4 mM 12C-IPA in the dark. In some cases, 10 μL of plasma was incubated with 280 μM, 560 μM, or 1.12 mM GHRP peptide in a total of 25 μL for 30 min. The bead supernatants were aspirated, and all samples were incubated with NuPAGE LDS sample buffer (Life Technologies) containing a further 4 mM 12C-IPA for 30 min at 60°C. Samples were resolved on SDS-PAGE at 150 V for 65 min before gels were stained with colloidal Coomassie (Sigma) and destained in 7% acetic acid and 10% methanol. An example Coomassie gel is shown in Figure S1. Fibrinogen bands were excised, diced into 1 mm3 slices, destained in 25 mM NH4HCO3 with 50% acetonitrile, dried in 100% acetonitrile, incubated with 40 mM dithiothreitol and washed. The gel slices were incubated with 5 mM 13C-IPA (Cambridge Isotopes) in 25 mM NH4HCO3, with 10% DMSO for 1 h at room temperature in the dark to alkylate the disulfide bond cysteines. Gel slices were washed and dried as above before digestion of proteins with 35 μL of 12.5 ng/μL trypsin (Promega, V5280, Trypsin Gold, Mass spectrometry Grade) in 25 mM NH4HCO3 overnight at 30°C. Peptides were eluted from the slices with 5% formic acid and 50% acetonitrile. Liquid chromatography, mass spectrometry and data analysis were performed as previously described (Al-Masri et al., 2023). Briefly, peptides were analyzed on a Thermo Fisher Scientific Ultimate 3000. Two hundred ng of peptide was injected and resolved on a 35 cm × 75 μm C18 reverse phase analytical column with integrated emitter using a 2 y-35% acetonitrile over 20 min with a flow rate of 250 nL/min. The peptides were ionized by electrospray ionization at +2.0 kV. Tandem mass spectrometry analysis was carried out on a Q-Exactive Plus mass spectrometer using HCD fragmentation. The data-dependent acquisition method acquired MS/MS spectra of the top 10 most abundant ions with charged states ≥2 at any one point during the gradient.", "projectTags" : [ ], "keywords" : [ "Human", "Disulfide bonds", "Fibrinogen" ], "doi" : "10.6019/PXD076459", "submissionType" : "COMPLETE", "publicationDate" : "2026-04-12", "updatedDate" : "2026-04-01", "submissionDate" : "2026-04-01", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Aster Pijning" ], "labPIs" : [ "Philip J. Hogg" ], "affiliations" : [ "School of Life Sciences, University of Technology Sydney, Faculty of Science, Sydney, New South Wales, Australia Centenary Institute, University of Sydney, Sydney, New South Wales, Australia" ], "instruments" : [ "Q Exactive" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "blood plasma", "Homo sapiens (Human)" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Blood plasma" ], "diseases" : [ ], "references" : [ "Pijning AE, Butera D, Hogg PJ. Multiple disulfide-bonded states confer extensive conformational diversity in fibrinogen. 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Understanding fundamental human NK cell metabolism provides opportunities for optimising NK cell therapies. Little is known about how glutamine, an important cell nutrient and carbon source, is utilised by human NK cells. To address this, we performed U13C-glutamine tracing experiments by Liquid Chromatography Mass Spectrometry (LCMS) and Gas Chromatography Mass Spectrometry (GCMS) analysis of human NK cells stimulated with IL-2 for 18 hours to provide a global overview of glutamine usage by these cells. Our results show that glutamine is taken up by resting NK cells and that this increases further upon IL-2 stimulation. Metabolite labelling analysis identified that IL-2 results in greater conversion of glutamine to glutamate, allowing for anaplerotic flux into the TCA cycle. The fate of the glutamine-derived carbons diverged at oxaloacetate (OAA) allowing both bioenergetic and biosynthetic outcomes - some carbons continued around the TCA cycle while others were exported, converted to aspartate and subsequently used for pyrimidine synthesis. Nucleotide synthesis by IL-2 activated NK cells was found to be essential for expression of the activation marker CD69. The data indicate that glutamine is a key nutrient taken up by human NK cells, and that IL-2 drives glutaminolysis. Subsequent glutamate is used to support the TCA cycle, generating energy and providing intermediates for de novo pyrimidine synthesis.", "dataProcessingProtocol" : "Raw DIA mass spectrometry data files were searched using Spectronaut (Biognosys) version 19. Raw MS files were searched against a mouse database (Swissprot Trembl, November 2023) with the following parameters: directDIA, false discovery rate set to 1%, protein N-terminal acetylation and methionine oxidation were set as variable modifications, and carbamidomethylation of cysteine residues was selected as a fixed modification. Perseus software (REF Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods13, 731–740 (2016)) was used to estimate protein copy numbers according to the method described in Wisniewski et al. (REF: Wiśniewski, J. R., Hein, M. Y., Cox, J. & Mann, M. A “proteomic ruler” for protein copy number and concentration estimation without spike-in standards. Mol. Cell. Proteomics 13, 3497–3506 (2014))", "sampleProcessingProtocol" : "Isolation of NK cells Peripheral blood mononuclear cells (PBMC) were isolated on the same day blood sample was drawn via density gradient centrifugation using Lymphoprep (Axis shield). Blood was combined with PBS at a ratio of 1:2, layered on top of lymphoprep (15ml) and centrifuged for 30 minutes at 300g on break 1. The buffy coat was extracted and washed in PBS (50ml). PBMC were counted and stained with Live/Dead viability stain, CD56 (clone HCD56/NCAM16.2) and CD3 (SK7/UCHT1) by staining in FACS buffer (PBS + 2% FC fetal calf serum (FCS)) for 20 mins on ice. Live CD56+CD3- NK cells were purified by flow sorting using a BD FACSAria™ III Cell Sorter. NK cell proteomics analysis: • Culture of NK cells for proteomic analysis Pure NK cells were seeded at 5x106 cells/ml in RPMI supplemented with 10% FCS and 1% penicillin/ streptomycin and stimulated with IL2 (500IU/ml). Cells were incubated at 37°C, 5% CO2 for 18 hours. NK cells were harvested, washed in ice cold PBS twice and NK cell pellets were snap frozen using liquid nitrogen. • Proteomic sample preparation Cells were lysed in 5% SDS and 50mM TEAB. Protein concentrations were determined using a micro-BCA assay. Aliquots containing 100 µg of protein were processed using the S-Trap mini protocol (Protifi) as recommended by the manufacturer, with minor modifications. Disulfide bonds were reduced with 20 mM DTT for 10 min at 95 °C, followed by alkylation with 40 mM IAA for 30 min in the dark. After loading the samples onto the S-Trap mini spin columns, the trapped proteins were washed five times with 500 µl of S-Trap binding buffer. Proteins were digested with trypsin (Pierce, Thermo Fisher) at a 1:40 enzyme-to-protein ratio in a two-step digestion: first overnight at 37 °C in 160 µl of 50 mM TEAB, and then for an additional 6 h at the same temperature. Peptides were eluted from the S-Trap mini columns by adding 160 µl of 50 mM TEAB, followed by 160 µl of 0.2% aqueous formic acid, and finally 160 µl of 50% acetonitrile containing 0.2% formic acid, with centrifugation at 1,000 × g for 1 min after each addition. Tryptic peptides were dried in a SpeedVac and stored at −20 °C. • Mass Spectrometry Peptides generated from NK cells were analyzed by Data-Independent Acquisition (DIA) on a Q-Exactive™ plus Mass Spectrometer (Thermo Scientific) coupled to a Dionex Ultimate 3000 RS (Thermo Scientific). LC buffers used are the following: buffer A (0.1% formic acid in Milli-Q water (v/v)) and buffer B (80% acetonitrile and 0.1% formic acid in Milli-Q water (v/v). A total of 1.5 µg of each sample was loaded at 10 μL/min onto a µPAC trapping C18 column (Pharmafluidics). The trapping column was washed for 6 min at the same flow rate with 0.1% TFA and then switched in-line with a Pharma Fluidics, 200 cm, µPAC nanoLC C18 column. The column was equilibrated at a flow rate of 300nl/min for 30 min. The peptides were eluted from the column at a constant flow rate of 300 nl/min with a linear gradient from 1% buffer B to 3.8% buffer B in 6 min, from 3.8% B to 12.5% buffer B in 40 min, from 12.5% buffer B to 41.3% buffer B within 176 min and then from 41.3 % buffer B to 61.3% buffer B in 14 min. The gradient is finally increased from 61.3% buffer B to 100% buffer B in 1 min, and the column was then washed at 100% buffer B for 10 min. Two blanks were run between each sample to reduce carry-over. The column was kept at a constant temperature of 50oC. Q-Exactive plus was operated positive ionization mode using an easy spray source. The source voltage was set to 2.2 Kv and the capillary temperature was 275oC. Data were acquired in DIA Mode. A scan cycle comprised a full MS scan (m/z range from 345-1155), resolution was set to 70,000, AGC target 3 x 106, maximum injection time 200 ms. MS survey scans were followed by DIA scans of dynamic window widths with an overlap of 0.5 Th. DIA spectra were recorded at a resolution of 17,500 at 200 m/z using an automatic gain control target of 3 x 106, a maximum injection time of 55 ms and a first fixed mass of 200 m/z. Normalised collision energy was set to 25 % with a default charge state set at 3. Data for both MS scan and MS/MS DIA scan events were acquired in profile mode. Proteomic sample preparation and mass spectrometry was conducted by the FingerPrints Proteomics facility at Dundee University.", "projectTags" : [ ], "keywords" : [ "Glutaminolysis", "Nk cells", "Il2" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-01", "updatedDate" : "2026-04-01", "submissionDate" : "2026-04-01", "downloadCount" : 26, "avgDownloadsPerFile" : 0.8965517, "botCount" : 0, "hubCount" : 26, "organicCount" : 0, "percentile" : 2, "yearlyDownloads" : [ { "year" : "2026", "count" : 26 } ], "submitters" : [ "Andrew Howden" ], "labPIs" : [ "Professor Clair Gardiner" ], "affiliations" : [ "Trinity College Dublin: Dublin, IE" ], "instruments" : [ "Q Exactive HF" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "blood" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Blood" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-independent acquisition" ], "sdrf" : "", "projectFileNames" : [ "AH-8-Rep.raw", "AH-7-Rep.raw", "AH-1.raw", "AH-23.raw", "AH-2.raw", "AH-6.raw", "AH-19-Rep.raw", "NK_cells_Report.csv", "AH-24.raw", "checksum.txt", "AH-18-Rep.raw", "AH-21.raw", "AH-3.raw", "AH-14-Rep.raw", "AH-4.raw", "AH-15-Rep.raw", "AH-25.raw", "AH-20.raw", "AH-17-Rep.raw", "NK_cells_Report.setup.txt", "AH-26.raw", "AH-16-Rep.raw", "AH-10-Rep.raw", "AH-5.raw", "AH-11-Rep.raw", "AH-22.raw", "AH-12-Rep.raw", "AH-9-Rep.raw", "AH-13-Rep.raw" ], "highlights" : { } }, { "accession" : "PXD076445", "title" : "ZO-1 phosphorylation upon ERK activation", "projectDescription" : "This dataset was generated to identify phosphorylation sites of ZO-1 regulated by ERK signaling. Cells were treated with TPA alone or with TPA in combination with the MEK inhibitor PD0325901, followed by phosphopeptide enrichment and LC–MS/MS analysis. TPA activates multiple kinases including ERK, whereas PD0325901 selectively inhibits MEK, the upstream activator of ERK. Thus, ERK activity is specifically suppressed in the co-treated condition. Phosphorylation sites detected in the TPA-treated samples but not in the TPA + PD0325901-treated samples are considered to be associated with ERK-dependent phosphorylation of ZO-1.", "dataProcessingProtocol" : "Raw LC–MS/MS data were processed using Proteome Discoverer (Thermo Fisher Scientific), and database searches were performed using Mascot (v2.8.1; Matrix Science) with an MS/MS ion search. The search database consisted of UniProtKB Canis lupus familiaris and a custom database including EGFP-ZO-1 and H2B-iRFP sequences. Trypsin was specified as the protease with up to two missed cleavages allowed. Carbamidomethylation of cysteine was set as a fixed modification, and oxidation (M) and phosphorylation (S, T) were set as variable modifications. Monoisotopic mass values were used, with a peptide mass tolerance of ±10 ppm and a fragment mass tolerance of ±0.8 Da. A false discovery rate (FDR) of <1% was applied at the peptide and protein levels. Label-free quantification was performed using equal amounts of input samples prior to phosphopeptide enrichment. No normalization based on total peptide amount was applied, as phosphopeptide enrichment alters peptide abundance.", "sampleProcessingProtocol" : "Cells were prepared as described above. Briefly, MDCK II ZO-1/2 dKO cells stably expressing EGFP-ZO-1 were cultured and treated with either 10 nM TPA or 10 nM TPA + 10 μM PD0325901 for 30 min. Cells were washed and lysed in HEPES-RIPA buffer containing protease and phosphatase inhibitors and Benzonase. Lysates were sonicated, centrifuged, and subjected to immunoprecipitation using GFP-Trap magnetic beads. After washing, bound proteins were digested on-bead using Trypsin/Lys-C at 37°C overnight. The resulting peptides were collected, and phosphopeptides were enriched using Phos-tag tips according to the manufacturer’s instructions. Peptides were reduced with DTT, alkylated with iodoacetamide, acidified with TFA, and desalted using ZipTip prior to LC–MS/MS analysis.", "projectTags" : [ ], "keywords" : [ "Phosphorylation", "Zo-1", "Erk" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-17", "updatedDate" : "2026-04-01", "submissionDate" : "2026-04-01", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Sayuki Hirano" ], "labPIs" : [ "Kazuhiro Aoki" ], "affiliations" : [ "Laboratory of Quantitative Biology, Department of Gene Mechanisms, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Japan" ], "instruments" : [ "LTQ Orbitrap Elite" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "kidney", "Canis familiaris (Dog) (Canis lupus familiaris)", "epithelial cell" ], "organisms" : [ "Canis familiaris (dog) (canis lupus familiaris)" ], "organismsPart" : [ "Epithelial cell", "Kidney" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "250109_hirano_Clupus_LG60_4_ZO1_TPA_MEKi.dat", "250109_hirano_Clupus_LG60_4_ZO1_TPA_MEKi.raw", "250109_hirano_Clupus_LG60_4_ZO1_TPA.raw", "250327_hirano_Clupus_LG60_4_2_ZO1_TPA_MEKi.raw", "250327_hirano_Clupus_LG60_4_2_ZO1_TPA_MEKi.dat", "250109_hirano_Clupus_LG60_4_ZO1_TPA.dat", "250327_hirano_Clupus_LG60_4_2_ZO1_TPA.raw", "250327_hirano_Clupus_LG60_4_2_ZO1_TPA.dat" ], "highlights" : { } }, { "accession" : "PXD076431", "title" : "Antifungal turbinmicin alters functional vesicle cargo", "projectDescription" : "Extracellular vesicle delivery in Candida biofilms is important for matrix delivery and associated drug-resistance. Prior study found the antifungal turbinmicin inhibits vesicle release. We find the antifungal turbinmicin inhibits both vesicle release as well as cargo loading. Using comparative vesicle proteome analysis in turbinmicin treated biofilms we identify several components blocked from cargo loading that play specific roles in the functional matrix protection mechanism.", "dataProcessingProtocol" : "Lumos acquired MS/MS data files were searched using Proteome Discoverer (ver. 2.5.0.400) Sequest HT search engine against Candida albicans SC5314 reference proteome (UP000000559; 09/09/2020 download with 6,030 protein entries). Static cysteine carbamidomethylation, and variable methionine oxidation plus asparagine and glutamine deamidation, 2 tryptic miss-cleavages and peptide mass tolerances set at 10 ppm with fragment mass at 0.6 Da were selected. Peptide and protein identifications were accepted under strict 1% FDR cut offs with high confidence XCorr thresholds of 1.9 for z=2 and 2.3 for z=3. Strict principles of parsimony were applied for protein grouping. Chromatograms were aligned for feature mapping and intensity-based quantification using unique and razor peptides. Normalization was performed on total peptide amount and scaling on all average. Protein abundance calculations were based on summed peptide abundances and background-based t-testing executed.", "sampleProcessingProtocol" : "Following vesicle isolation, an identical number of vesicles from control and treatment groups were subjected to enzymatic “in liquid” digestion and mass spectrometric analysis was done at the Mass Spectrometry Facility, Biotechnology Center, University of Wisconsin–Madison. Proteins were isolated from freshly obtained vesicles via methanol:chloroform:water extraction assisted by sonication as follows. Two aliquots of 200μl solution of vesicles in storage buffer were diluted with 600μl of methanol and sonicated in a sonication bath for 2 minutes. 200ul of chloroform was added next and vortexed well before adding 400μl of water followed by another round of vortexing and 1-minute spin at room temperature (max speed). Upper layer was removed and 800μl of acetone was added, vortexed and spun and supernatant removed. Additional rinse with 800μl methanol followed as above and the pellets were dried. Two pellets per sample were resolubilized and denatured in 20μl of 8M Urea in 50mM NH4HCO3 (pH8.5) for 10 min then pooled together. 10μl was taken for BCA protein readings and 20μl was diluted to 60μl for reduction step with: 2.5μl of 25mM DTT and 37.5μl of 25mM NH4HCO3 (pH8.5). Incubated at 56°C for 15 minutes, cooled on ice to room temperature then 3μl of 55mM CAA (chloroacetaminde) was added for alkylation and incubated in darkness at room temperature for 15 minutes. Reaction was quenched by adding 8μl of 25mM DTT. Finally, 4μl of Trypsin/LysC solution [100ng/μl 1:1 Trypsin (Promega) and LysC (FujiFilm) mix in 25mM NH4HCO3] and 25μl of 25mM NH4HCO3 (pH8.5) was added to 100µl final volume. Digestion was conducted for 2 hours at 42°C then additional 2µl of trypsin/LysC mix was added and digestion proceeded o/n at 37°C. Reaction was terminated by acidification with 2.5% TFA [Trifluoroacetic Acid] to 0.3% final. Digested and acidified samples were cleaned up using OMIX C18 SPE cartridges (Agilent) per manufacturer protocol and eluted in 20µl of 60/40/0.1% ACN/H2O/TFA, dried to completion in the speed-vac and finally reconstituted in 10µl of 0.1% formic acid. Peptides were analyzed by Orbitrap Fusion™ Lumos™ Tribrid™ platform, where 2µl were injected using Dionex UltiMate™3000 RSLCnano delivery system (ThermoFisher Scientific) equipped with an EASY-Spray™ electrospray source (held at constant 50°C). Chromatography of peptides prior to mass spectral analysis was accomplished using capillary emitter column (PepMap® C18, 2µM, 100Å, 500 x 0.075mm, Thermo Fisher Scientific). NanoHPLC system delivered solvents A: 0.1% (v/v) formic acid , and B: 80% (v/v) acetonitrile, 0.1% (v/v) formic acid at 0.30 µL/min to load the peptides at 2% (v/v) B, followed by quick 2 minute gradient to 5% (v/v) B and gradual analytical gradient from 5% (v/v) B to 37.5% (v/v) B over 73 minutes when it concluded with rapid ramp to 95% (v/v) B for a 5minute flash-out. As peptides eluted from the HPLC-column/electrospray source survey MS scans were acquired in the Orbitrap with a resolution of 120,000 followed by HCD-type MS2 fragmentation into Ion Trap (32% collision energy) under ddMSnScan 1 second cycle time mode with peptides detected in the MS1 scan from 350 to 1600 m/z; redundancy was limited by dynamic exclusion and MIPS filter mode ON.", "projectTags" : [ ], "keywords" : [ "Candida", "Proteomics", "Extracellular vesicles", "Turbinmicin" ], "doi" : "10.6019/PXD076431", "submissionType" : "COMPLETE", "publicationDate" : "2026-04-06", "updatedDate" : "2026-04-01", "submissionDate" : "2026-04-01", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Greg Sabat" ], "labPIs" : [ "Dr. David R. Andes" ], "affiliations" : [ "Department of Medicine, University of Wisconsin - Madison" ], "instruments" : [ "Orbitrap Fusion Lumos" ], "softwares" : [ ], "quantificationMethods" : [ "Relative quantification" ], "sampleAttributes" : [ "vegetative cell (sensu Fungi)", "Candida albicans (Yeast)", "disease free" ], "organisms" : [ "Candida albicans (yeast)" ], "organismsPart" : [ "Vegetative cell (sensu fungi)" ], "diseases" : [ "Disease free" ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "200413_Vesicles-4_Andes.raw", "200413_Vesicles-3_Andes.raw", "200413_Vesicles-2_Andes.raw", "200413_Vesicles-1_Andes.raw", "200413_Vesicles_Andes.mzML", "UP559_C_albicans_SC5314_090920.fasta", "200413_Vesicles_Andes.mzid" ], "highlights" : { } }, { "accession" : "PXD076398", "title" : "Proteomic analysis reveals shared biological pathways linking acrolein to biomolecular changes in the acute phase of rat spinal cord injury", "projectDescription" : "Spinal cord injury (SCI) is a devastating and highly heterogeneous condition that often leads to permanent sensory, motor, and autonomic impairments. Beyond the initial mechanical trauma, a cascade of secondary biochemical events further amplifies tissue damage and functional loss. In this study, we investigate the proteomic consequences of the reactive aldehyde acrolein within the context of secondary injury pathology. Using comparative proteomic analyses, we evaluate biomolecular changes in SCI vs sham injury and acrolein vs saline injection models. We then apply integrated computational approaches to identify shared protein alterations and biological pathways that may mechanistically link acrolein exposure to SCI pathology.", "dataProcessingProtocol" : "Data processing was performed by MtoZ Biolabs. Mass spectrometry (MS) data was acquired using an Exploris 480 mass spectrometer. Peptides were identified from the raw mass spectrometry data with Data-Independent Acquisition – Neural Networks (DIANN, v1.9) software using the UP000002494_10116.fasta. database.", "sampleProcessingProtocol" : "Male Sprague Dawley rats were used for this study. Spinal cord samples from four different conditions (SCI, sham, acrolein injection, and saline injection) were collected 24 hours post-procedure (n = 5/group) and sent to MtoZ Biolabs for proteomic processing. All rats underwent a T10 laminectomy. The SCI group received a moderate contusive injury using a Horizon Impactor (force = 200 kDy). The sham group received a laminectomy without injury. The acrolein and saline injection groups received either acrolein or saline injected directly into the spinal cord.", "projectTags" : [ ], "keywords" : [ "Acrolein", "Rat", "Proteomics", "Spinal cord injury" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-02", "updatedDate" : "2026-03-31", "submissionDate" : "2026-03-31", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Rachel Stingel" ], "labPIs" : [ "Riyi Shi" ], "affiliations" : [ "1. Center for Paralysis Research, 2. Department of Basic Medical Sciences, 3. College of Veterinary Medicine, 4. Weldon School of Biomedical Engineering, 5. Purdue University, West Lafayette, IN 47907" ], "instruments" : [ "orbitrap" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Rattus norvegicus (Rat)", "spinal column", "thoracic spine" ], "organisms" : [ "Rattus norvegicus (rat)" ], "organismsPart" : [ "Thoracic spine", "Spinal column" ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.64898/2026.03.11.711153" ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "UP000002494_10116.fasta.gz", "checksum.txt", "Acrolein-Protein.xlsx", "SCI-sham_RAW.zip", "SCI-Protein.xlsx", "Acrolein-Peptide.xlsx", "SCI-Peptide.xlsx", "Acrolein-saline_RAW.zip" ], "highlights" : { } }, { "accession" : "PXD076296", "title" : "Zero-Shot De Novo Peptide Sequencing with Open Post-Translational Modification Discovery", "projectDescription" : "Proteins play essential roles in biology, yet identifying their precise sequences and modifications remains challenging. De novo peptide sequencing offers a powerful solution by directly inferring sequences from mass spectrometry data without relying on protein databases. Recent deep learning models have significantly advanced this task but remain trapped in a major dilemma: they require labeled training data to recognize post-translational modifications (PTMs), which is unavailable for most biologically relevant but rare or unknown modifications. We solve this long-standing problem by introducing RNovA, a transformer-based de novo sequencing algorithm enhanced with relative positional embeddings and a reinforcement-learning–style sequential decision framework. RNovA enables open PTM discovery in a zero-shot settingwithout retraining or a predefined list of candidate residues—while maintaining state-of-the-art performance on standard benchmarks. Demonstrating this capability, we successfully identified peptides modified by kynurenine—an uncommon and biologically relevant PTM—in clinical samples from rheumatoid arthritis patients. RNovA overcomes key limitations of existing methods and provides a foundation for exploring previously inaccessible regions of the proteome, including peptides with unexpected or unannotated modifications. This capability is widely needed in immunology, biomarker discovery, and biomedical research.", "dataProcessingProtocol" : "Both PathSearcher and SeqFiller are trained within an episodic, value-based reinforcementlearning–style framework, framing de novo peptide sequencing as a sequential decision-making problem. At each decoding iteration, the model predicts action-value scores (Q-values) over nodes (PathSearcher) or candidate amino acids (SeqFiller), and actions are sampled from a Boltzmann policy induced by the predicted Q-values. 18 Submitted Manuscript: Confidential Template revised July 2024 550 555 560 565 570 575 580 585 590 In PathSearcher, training proceeds by generating paths that start at the origin node (0 Da), traverse the spectrum graph to the precursor mass node, and then return in reverse, with the number of bidirectional passes specified by the user. For each decoding step, an oracle actionvalue label \uD835\uDC44 * ( \uD835\uDC60 , \uD835\uDC4E ) ∈ { 0 , 1 } is defined to indicate whether taking action \uD835\uDC4E at the current partial path state \uD835\uDC60 remains consistent with the ground-truth optimal fragmentation path and can still lead to an exact round-trip match (38). Importantly, once any incorrect decision is made, the decoding trajectory becomes irrecoverable: for any subsequent state \uD835\uDC60that contains an error, * = \uD835\uDC44 ( \uD835\uDC60 , \uD835\uDC4E ) 0 for all actions \uD835\uDC4E. SeqFiller follows an analogous procedure, where oracle actionvalue labels indicate whether extending the current partial peptide sequence with a given amino acid remains consistent with the ground-truth target sequence. This structure enforces sequencelevel consistency and avoids token-level teacher forcing. Model parameters are optimized using binary cross-entropy loss over these step-wise oracle action-value labels, rather than token-level or residue-level supervision. This formulation allows the models to learn from global, sequence-level consistency signals and to tolerate ambiguity arising from missing fragment ions or unknown modifications. The resulting supervision structure and its sparsity are illustrated in Figure S4. Because oracle action-value labels are defined with respect to complete decoding trajectories, training requires full-sequence exploration before supervision can be applied. The inference strategy used at test time is identical to that used during training, ensuring consistency between learning and application. The models are trained on the MassiveKB (39) dataset, which includes peptides with various modifications such as acetylation, phosphorylation, carbamidomethylation, and multiple isotopic and labeling tags. The only preprocessing step involves lossy compression of the input spectra, where m/z values are rounded to a tolerance 10 -4 of and intensities to within 1%. This step was performed solely to reduce storage and memory overhead during training on the large-scale MassiveKB dataset and is not required when applying RNovA in practical scenarios.", "sampleProcessingProtocol" : "Synthetic Peptides with 293T Cells Protein extraction and in-solution digestion of 293T cells followed the same protocol as for yeast. After reconstituting the peptides to a concentration of 0.5 µg/µL, synthetic peptides were spiked into the 293T peptide mixture at a final concentration of 400 nM for mass spectrometry analysis. To further evaluate the sensitivity of RNovA for PTM identification, we additionally performed the same spiking experiment using a dilution series of 400 fmol, 40 fmol, and 4 fmol, while keeping all other experimental conditions unchanged. Peptides were analyzed on the same Orbitrap Ascend Tribrid instrument operated in DDA mode with a 120-minute LC gradient. RNovA analysis was performed using 10 decoding iterations with a beam size of 1 (greedy decoding), and results were filtered at 1% FDR using the same procedure as in other benchmarking experiments. For database search–based validation, open-modification searches were conducted using MSFragger v4.3 within the FragPipe framework. The FragPipe “Open Search” workflow was run with its default settings; key parameters include a precursor mass tolerance of –150 to +500 Da, a fragment mass tolerance of 20 ppm, and result filtering at 1% FDR. Preparation of RA synovial tissue samples and LC–MS/MS acquisition Synovial tissue samples were collected from rheumatoid arthritis (RA) patients with appropriate ethical approval. Proteins were extracted from the tissue using a standard bottom-up proteomics workflow. The samples were subjected to reduction and alkylation, followed by enzymatic digestion with trypsin to generate peptide fragments. Peptides were then analyzed using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Chromatographic separation was performed with a 360-minute gradient on a reversed-phase column, and the resulting eluate was analyzed on an Orbitrap Eclipse mass spectrometer operating in data-dependent acquisition (DDA) mode.", "projectTags" : [ ], "keywords" : [ "Deep learning", "Ptm", "De novo" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-29", "updatedDate" : "2026-03-29", "submissionDate" : "2026-03-29", "downloadCount" : 2, "avgDownloadsPerFile" : 0.017094018, "botCount" : 0, "hubCount" : 2, "organicCount" : 0, "percentile" : 0, "yearlyDownloads" : [ { "year" : "2026", "count" : 2 } ], "submitters" : [ "Zeping Mao" ], "labPIs" : [ "Ming Li" ], "affiliations" : [ "Canada Research Chair in Bioinformatics University Professor David R. Cheriton School of Computer Science University of Waterloo, Ontario, Canada" ], "instruments" : [ "Orbitrap Fusion Lumos", "orbitrap", "Orbitrap Fusion" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "arthritis", "cell culture", "Homo sapiens (Human)", "permanent cell line cell", "Saccharomyces cerevisiae (Baker's yeast)" ], "organisms" : [ "Homo sapiens (human)", "Saccharomyces cerevisiae (baker's yeast)" ], "organismsPart" : [ "Cell culture", "Permanent cell line cell" ], "diseases" : [ "Arthritis" ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "20250427-MZP-293T-400nM-Group2_calibrated_rnova_denovo_seq.csv", "117_DDA_1ug_360min_proteome.raw", "20250427-MZP-yeast-no_label-0_calibrated_rnova_denovo_path.csv", "A12E_360MIN.raw", "20250912-group1-40fmol_calibrated_rnova_denovo_path.csv", "20250427-MZP-yeast-no_label-1_calibrated_rnova_denovo_path_001fdr.csv", 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"20250912-group2-40fmol_calibrated_rnova_denovo_path_001fdr.csv", "20250427-MZP-293T-400nM-Group2_calibrated_rnova_denovo_seq_001fdr.csv", "20250912-group3-4fmol_calibrated_rnova_denovo_path.csv", "119_DDA_1ug_360min_proteome_calibrated_rnova_denovo_path.csv", "120_prm_500ng_70min_kyn_20250427205600_rnova_denovo_seq.csv", "A1232E_360min_2.raw", "20250912-group3-40fmol_calibrated_rnova_denovo_seq_001fdr.csv", "20250912-group1-400fmol_calibrated_rnova_denovo_seq.csv", "20250912-group1-4fmol_calibrated_rnova_denovo_path_001fdr.csv", "120_DDA_1ug_360min_proteome_calibrated_rnova_denovo_path.csv", "20250427-MZP-yeast-no_label-0_calibrated_rnova_denovo_path_001fdr.csv", "20250427-MZP-293T-400nM-Group3_calibrated_rnova_denovo_seq_001fdr.csv", "A1232E_360min_1decoy_random0.40_rnova_denovo_path_001fdr.csv", "20250427-MZP-yeast-no_label-2.raw", "A1232E_360min_2decoy_random0.40_rnova_denovo_path_001fdr.csv", "20250427-MZP-yeast-no_label-3_calibrated_rnova_denovo_path.csv", "20250912-group3-4fmol.raw", "20250912-group2-400fmol_calibrated_rnova_denovo_path_001fdr.csv", "119_DDA_1ug_360min_proteome_calibrated_rnova_denovo_seq.csv", "20250427-MZP-293T-400nM-Group3.raw" ], "highlights" : { } }, { "accession" : "PXD076216", "title" : "Proteomic Signatures in Cerebrospinal Fluid and Their Clinical Associations in Patients with ME/CFS", "projectDescription" : "This study evaluated the cerebrospinal fluid (CSF) proteomes from 31 patients diagnosed with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We quantified 902 proteins, each expressed in at least eleven samples, and systematically categorized clinical factors relevant to ME/CFS symptoms—including autonomic dysfunction, neuroinflammation and metabolic disturbances.", "dataProcessingProtocol" : "Raw spectra were processed using MaxQuant (v2.2.0.0) and the Andromeda search engine against the UniProt human FASTA database (June 2022). Search parameters included a 4.5 ppm tolerance for precursor ions and 0.5 Da for MS/MS fragments. Trypsin was specified as the digestion enzyme, permitting up to two missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification, while methionine oxidation and N-terminal acetylation were set as variable modifications. Protein identifications were filtered at a 1% FDR.", "sampleProcessingProtocol" : "CSF proteomic profiling was conducted using a QExactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to an EASY-nLC 1000 liquid chromatography system. Peptides were separated using a C18 pre-column (2 cm, 100 μm ID) and analytical column (10 cm, 75 μm ID). A 150-minute linear gradient elution from 4% to 100% acetonitrile was applied at 250 nL/min. Mass spectra were acquired in positive ion mode over the m/z range of 400–1750 with a resolution of 70,000. The top 10 ions were subjected to higher-energy collisional dissociation (HCD) at 25% normalized energy, with fragment spectra collected at 17,500 resolution.", "projectTags" : [ ], "keywords" : [ "Me/cfs", "Proteomics", "Lc-ms/ms", "Cerebrospinal fluid", "Myalgic encephalomyelitis", "Chronic fatigue syndrome" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-05", "updatedDate" : "2026-03-27", "submissionDate" : "2026-03-27", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Peng Li" ], "labPIs" : [ "Jonas Bergquist" ], "affiliations" : [ "Analytical Chemistry and Neurochemistry, Department of Chemistry - BMC, Uppsala University, Uppsala, Sweden; The ME/CFS Collaborative Research Centre at Uppsala University, Uppsala, Sweden;" ], "instruments" : [ "Q Exactive" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "cerebrospinal fluid", "chronic fatigue syndrome" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Cerebrospinal fluid" ], "diseases" : [ "Chronic fatigue syndrome" ], "references" : [ "Bragée B, Li P, Meadows D, Widgren A, Sjögren P, Ghatan PH, Bertilson BC, Xiao W, Bergquist J. Proteomic signatures in cerebrospinal fluid and their clinical associations in patients with ME/CFS. Sci Rep. 2026--pubMed:41932997--doi: 10.1038/s41598-026-46965-1" ], "experimentTypes" : [ "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "proteinGroups.txt", "P09_moderate_NoPOTS.raw", "P22_severe_POTS.raw", "P11_moderate_POTS.raw", "P18_mild_NoPOTS.raw", "P12_moderate_NoPOTS.raw", "P02_severe_NoPOTS.raw", "P26_moderate_NoPOTS.raw", "P28_severe_NoPOTS.raw", "checksum.txt", "P24_moderate_NoPOTS.raw", "P13_moderate_NoPOTS.raw", "P10_moderate_NoPOTS.raw", "P07_moderate_NoPOTS.raw", "P06_moderate_POTS.raw", "P01_severe_POTS.raw", "peptides.txt", "P23_mild_POTS.raw", "P15_moderate_POTS.raw", "P05_moderate_NoPOTS.raw", "P29_severe_POTS.raw", "P16_moderate_NoPOTS.raw", "evidence.txt", "P27_moderate_NoPOTS.raw", "P31_moderate_NoPOTS.raw", "P20_moderate_NA.raw", "P04_moderate_POTS.raw", "P14_moderate_NoPOTS.raw", "P17_severe_NoPOTS.raw", "sdrf.tsv", "P19_moderate_NoPOTS.raw", "P08_moderate_NoPOTS.raw", "P25_moderate_NoPOTS.raw", "P21_severe_POTS.raw", "P03_moderate_NoPOTS.raw", "P30_mild_NoPOTS.raw" ], "highlights" : { } }, { "accession" : "PXD076266", "title" : "Protein propionylation in Pcca deficiency", "projectDescription" : "To determine the prevalence and identify any enrichment of lysine propionylation in PCC deficiency, an analysis comparing the propionylated liver proteins of control and liver-specific Pcca knockout mice was performed. Liver-specific knockout of PCC activity was induced in mice with floxed axons for the Pcca gene (Pcca ff) at 5 weeks of age using iCre-AAV with a TBG promoter. For controls, Pcca ff mice were injected with eGFP-AAV under the same promoter.", "dataProcessingProtocol" : "A Maxquant (2.1) software was used to analyze and search the 8 raw MS files against the Mus musculus protein database. The search parameters were set as follows: the protein modifications were carbamidomethylation (C) (fixed), oxidation (M) (variable), propionylation (K) (variable); the enzyme specificity was set to trypsin; the maximum missed cleavages were set to 2; the precursor ion mass tolerance was set to 20 ppm, and MS/MS tolerance was 0.05 Da. Raw signals were translated using log2 transformation, and data normalization was performed using the limma R package (v3.46.0). A 2-tailed Student’s t-test was used for differential analysis with a p-value of 0.05 and a fold change of 2.0 to identify differential proteins.", "sampleProcessingProtocol" : "Livers of control and Pcca KO mice were collected, flash frozen, and sent for analysis at Creative Proteomics. Four samples from each group were digested by trypsin. Anti-propionyl-lysine antibody beads were used to enrich propionyl peptides, which were then analyzed by NanoLC-MS/MS and identified using an LTQ Orbitrap ETD MS platform.", "projectTags" : [ ], "keywords" : [ "Mouse", "Liver", "Nano lc-ms/ms" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-09", "updatedDate" : "2026-03-27", "submissionDate" : "2026-03-27", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Jasmine Encarnacion" ], "labPIs" : [ "Michael J Wolfgang" ], "affiliations" : [ "Department of Physiology, Pharmacology and Therapeutics, The Johns Hopkins University School of Medicine, Baltimore, Maryland." ], "instruments" : [ "LTQ Orbitrap XL ETD" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "propionic acidemia", "liver", "Mus musculus (Mouse)" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Liver" ], "diseases" : [ "Propionic acidemia" ], "references" : [ ], "experimentTypes" : [ "Affinity purification coupled with mass spectrometry proteomics" ], "sdrf" : "", "projectFileNames" : [ "ff_iCre964-1.raw", "checksum.txt", "ff_eGFP961-3.raw", "ff_eGFP963-1.raw", "ff_eGFP961-2.raw", "Analytical_Service_Report_for_PropionylLysine-proteomics_Analysis_CPJS02102505.pdf", "ff_iCre963-3.raw", "Dataresult_CPJS02102505.xlsx", "Description_for_Excel_file_CPJS02102505.pdf", "ff_iCre963-2.raw", "ff_eGFP961-4.raw", "ff_iCre964-3.raw" ], "highlights" : { } }, { "accession" : "PXD076268", "title" : "Phosphorylation of rhizobial effector NopZ by soybean NORK promotes association with NENA and enhances nodulation", "projectDescription" : "Legume-rhizobial symbiosis, involving the intracellular accommodation of rhizobia within host cells for nitrogen fixation, represents a unique model among plant-microbe interactions. While this symbiosis requires sophisticated regulatory networks, direct host control over symbiont proteins remains largely elusive. Here, we demonstrate that soybean Nodulation Receptor Kinase (GmNORK) interacts with and directly phosphorylates the Sinorhizobium fredii HH103 effector NopZ. This host-mediated phosphorylation promotes NopZ nuclear/perinuclear enrichment and is associated with enhanced NopZ interaction with the nucleoporin GmNENA at the nuclear envelope/nuclear pore complex, establishing a key signaling step that links membrane-proximal symbiotic signaling to the nuclear pore. Expression of phosphomimetic NopZ variants enhances soybean nodulation, whereas GmNENA-silencing in soybean roots reduces nodule numbers. Our findings reveal a previously unknown model for plant-microbe interactions, where the direct phosphorylation of a bacterial effector by a host receptor kinase provides an essential regulatory mechanism to direct effector localization and promote the symbiotic program.", "dataProcessingProtocol" : "The resulting peptides were analyzed by LC–MS/MS using a Q Exactive HF mass spectrometer. Raw data were searched against a custom database containing the sequences of the target proteins along with the E. coli proteome. This dataset was used to identify protein modifications and modification sites under co-expression conditions", "sampleProcessingProtocol" : "For MS, His-GmNORKαCD-Myc was co-expressed with His-NopZ-FLAG by using Duet vectors in E. coli cells. Total proteins were extracted from cells. Samples were incubated on ice for 30 min and centrifuged three times at 12,000 × g for 10 min at 4°C. The supernatant was added to 10 μL Ni-NTA agarose beads followed by end-to-end rotation at 4°C for 4 h. The beads were washed at least 15 times with washing buffer. The bound proteins were then eluted using imidazole-containing buffer and separated by SDS–PAGE. Protein bands corresponding to the expected molecular weight were excised from the gel and subjected to in-gel digestion following reduction and alkylation. The resulting peptides were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) to examine the modification status of His-tagged proteins under co-expression conditions.", "projectTags" : [ ], "keywords" : [ "Soybean;phosphorylation;effector;npc" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-10", "updatedDate" : "2026-03-27", "submissionDate" : "2026-03-27", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Yutao Lei" ], "labPIs" : [ "Yangrong Cao" ], "affiliations" : [ "National Key Lab of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University" ], "instruments" : [ "Q Exactive HF" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Escherichia coli" ], "organisms" : [ "Escherichia coli" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "20210514_Z-1.index", "20210514_Z-1.raw" ], "highlights" : { } }, { "accession" : "PXD076239", "title" : "F-actin dynamics couples sphingolipid metabolism to epithelial barrier integrity in chronic colitis (proteomics)", "projectDescription" : "Intestinal barrier dysfunction is a hallmark of gastrointestinal disorders, including inflammatory bowel diseases (IBD). In IBD, the disruption of the gut barrier causes liquid loss and persistent immune response. Understanding the cellular events underlying epithelial barrier disruption during chronic inflammation is essential for targeting increased intestinal permeability. Filamentous actin (F-actin) destabilization accompanied by metabolic dysfunction has been previously reported in the Muc2 knockout colitis model; however, the mechanistic contribution of these defects to chronic intestinal barrier loss remains unclear. Here, we identify impaired cytoskeleton dynamics as a critical driver of intestinal barrier dysfunction in chronic colitis. An imbalance between polymeric and monomeric actin disrupts the epithelial barrier in both 3D organoid system and in vivo. Actin and associated factors were identified as primary interactors of claudin-3, and this interaction was reduced under chronic inflammatory conditions in vivo. Further analysis revealed ceramide metabolism as a potential metabolic regulator of actin dynamics and barrier integrity during chronic inflammation. Consistently, intestinal samples from IBD patients showed concurrent disruption of tight and adherens junctions and reduced F-actin levels. Together, these findings reveal F-actin dynamics as one of the key mechanisms of barrier dysfunction in IBD and highlight ceramide metabolism as a potential therapeutic target in IBD.", "dataProcessingProtocol" : "Intensity-Based Absolute Quantification (iBAQ) scores for proteins identified in the mock IP were subtracted from the corresponding values from the IPs from Muc2 KO mice and the control animals. Next, protein lists were depleted for proteins annotated to heat shock proteins, immunoglobulins, keratins, ribosomal proteins and elongation factors as these proteins are common nonspecific contaminants in immunoprecipitation experiments and are unlikely to be related to tight junctions. The remaining protein lists were subjected to functional annotation (gene ontology, GO) using clusterProfiler [68] and org.Mm.eg.db [69] R packages based on “Biological process” ontology.", "sampleProcessingProtocol" : "Colonic tissues were collected from Muc2+/+ and Muc2 KO mice (n = 6 per group) and immediately homogenized in ice-cold lysis buffer containing 25 mM HEPES (pH 8.0), 200 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, supplemented with 1 mM sodium metabisulfite (NAMBS) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Homogenization was performed using a glass homogenizer with a tight pestle. Lysates were centrifugated at 13,000×g for 30 min at 4℃, and the supernatants from each genotype were combined for immunoprecipitation. For each genotype, 2.5 mg of total protein extract was incubated with 50 µL of anti-claudin-3 antibody (#ab15102, Abcam, UK) and Protein A Sepharose beads (70-80 µL packed volume). Binding reactions were carried out in 25 mM HEPES (pH 8.0), 200 mM NaCl, 5 mM EDTA, 0.1% NP-40, 1 mM NAMBS, and 1 mM PMSF for 4 h at 4℃. Beads were washed three times with a wash buffer (25 mM HEPES, pH 8.0, 500 mM NaCl, 5 mM EDTA, 0.1% NP-40, 1 mM NAMBS, 1 mM PMSF). Protein complexes were eluted in citrate buffer (pH 3.0), and eluates were immediately neutralized. Protein precipitation was performed using the trichloroacetic acid / deoxycholate (TCA/DOC) method. Pellets were resuspended in 35 µL of Laemmli sample buffer and heat-denatured prior to proteomic analysis. A mock IP control was performed using non-functional mock antibodies (verified to show no reactivity with colonic lysates in Western blot). For this control, a 1:1 mixture of protein extracts from Muc2 KO mice and their wilt-type littermates was used. Denatured samples were shipped to BGI (Hong Kong, China) for LC-MS/MS analysis and peptide annotation.", "projectTags" : [ ], "keywords" : [ "Cldn3", "Actin", "Ibd" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-01", "updatedDate" : "2026-03-27", "submissionDate" : "2026-03-27", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Snezhanna Medvedeva" ], "labPIs" : [ "Elena Kozhevnikova" ], "affiliations" : [ "Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences" ], "instruments" : [ "Q Exactive HF" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "colon", "epithelial cell", "Mus musculus (Mouse)", "bowel dysfunction" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Epithelial cell", "Colon" ], "diseases" : [ "Bowel dysfunction" ], "references" : [ "null--pubMed:0--doi: 10.64898/2026.01.21.700747" ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "23P06840002.mgf", "23P06840003.mgf", "23P06840001.mgf", "ENK2CONT.dat", "23P06840001.raw", "ENK1MUT.dat", "ENK3MOCK.dat", "23P06840002.raw", "23P06840003.raw" ], "highlights" : { } }, { "accession" : "PXD076189", "title" : "Structural Basis of Membrane Potential Coupled Vectorial CO₂ Hydration by the DAB2 Complex in Chemolithoautotrophs", "projectDescription" : "The fixation of dissolved inorganic carbon (DIC) such as CO2 and bicarbonate is fundamental to the global primary production. Many autotrophs depend on a diversity of CO2-concentrating mechanisms (CCMs) to overcome the inefficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase and the limited supply of DIC. While cyanobacterial CCMs are well characterized, analogous systems in chemolithoautotrophs, specifically active DIC uptake systems have long been overlooked. Here, we present the first cryo-EM analysis of DAB2, an essential membrane-associated complex for CO₂ uptake in Halothiobacillus neapolitanus. The cytoplasmic subunit DabA2 displays a β-carbonic anhydrase-like fold, while the transmembrane subunit DabB2 resembles the proton-conducting subunits of respiratory Complex I. Purified DAB2 binds CO₂ independent of proton motive force (PMF) however, did not spontaneously hydrate CO2. Structural analysis reveals a deeply buried active site only accessible via gated substrate tunnels, suggesting substrate access and catalysis are tightly regulated. The transmembrane helix of DabA2 forms the proton pathway and potentially couples proton translocation to the catalysis. These features define a vectorial CO2 hydration mechanism that prohibits reverse bicarbonate dehydration. Our findings establish DAB2 as a prototype of a previously unrecognized family of PMF-driven carbonic anhydrases, elucidating a novel strategy for CO₂ capture in non-photosynthetic autotrophs.", "dataProcessingProtocol" : "Analysis of DIA data was performed using the DIA-NN version 1.9 (ref.58) using a uniprot protein database from E.coli BL21 including target proteins to generate a data set specific spectral library for the DIA analysis. The neural network based DIA-NN suite performed noise interference correction (mass correction, RT prediction and precursor/fragment co-elution correlation) and peptide precursor signal extraction of the DIA-NN raw data. The following parameters were used: Full tryptic digest was allowed with two missed cleavage sites, and oxidized methionines (variable) and carbamidomethylated cysteins (fixed). Match between runs and remove likely interferences were enabled. The precursor FDR was set to 1%. The neural network classifier was set to the single-pass mode, and protein inference was based on genes. Quantification strategy was set to any LC (high accuracy). Cross-run normalization was set to RT-dependent. Library generation was set to smart profiling. DIA-NN outputs were further evaluated using the SafeQuant script59,60 modified to process DIA-NN outputs", "sampleProcessingProtocol" : "Bacteria were cultivated as described in the complementation assay above (n = 4 biological replicates). The cell pellets were resuspended in 300 μl lysis buffer (2% sodium lauroyl sarcosinate (SLS), 100 mM ammonium bicarbonate) and heated at 90°C for 40 min. The protein amount was determined by bicinchoninic acid protein assay (Thermo Scientific). Proteins were incubated with 5 mM Tris(2-carboxyethyl) phosphine (Thermo Fischer Scientific) and 10 mM Chloroacetamide at 90°C for 15 min (Sigma Aldrich) and further processed with SP3 (ref.57). Proteins were bound to 4 µl SP3 beads (40% v/v bead stock) in presence of 70 % acetonitrile for 15 min at room temperature, followed by two washes of beads with 70 % ethanol and an additional wash with acetonitrile. After removal of the supernatant, 1 µg trypsin in 100 mM NH4HCO3 was added to the beads and digested shaking overnight at 30°C. Digested proteins were harvested and purified using C18 solid phase extraction. Peptides were finally dried, reconstituted in 0.1 % trifluoroacetic acid (TFA) and analyzed using liquid-chromatography-mass spectrometry carried out on an Exploris 480 instrument connected to an VanquishNeo and a nanospray flex ion source (all Thermo Scientific). Peptide separation was performed on a reverse phase HPLC column (75 μm x 26 cm) packed in-house with C18 resin (1.9 μm Reprosil-AQ; Dr. Maisch). The following separating gradient was used: 100% solvent A (0.1% formic acid) to 40% solvent B (99.85% acetonitrile, 0.15% formic acid) over 32 minutes at a flow rate of 300 nl/min. The direct injection setup was applied. MS raw data was acquired in data independent acquisition (DIA) mode. The funnel RF level was set to 40. Full MS resolution was set to 120.000 at m/z 200. AGC target value for fragment spectra was set at 3000%. 45 windows of 14 Da were used with an overlap of 1 Da between m/z 320-950. Resolution was set to 15,000 and IT to 22 ms. Stepped HCD collision energy of 25, 27.5, 30 % was used. MS1 data was acquired in profile, MS2 DIA data in centroid mode.", "projectTags" : [ ], "keywords" : [ "Co2", "Halothiobacillus neapolitanus" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-17", "updatedDate" : "2026-03-26", "submissionDate" : "2026-03-26", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Timo Glatter" ], "labPIs" : [ "Timo Glatter" ], "affiliations" : [ "Max Planck Institute for Terrestrial Microbiology Karl-von-Frisch Str. 10 35043 Marburg Germany" ], "instruments" : [ "Q Exactive HF" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Escherichia coli" ], "organisms" : [ "Escherichia coli" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "Dab2-1-34.raw", "Dab2-1-69.raw", "Dab2-1-77.raw", "Dab2-1-26.raw", "Dab2-1-51.raw", "Dab2-1-43.raw", "Dab2-1-17.raw", "Dab2-1-9.raw", "Dab2-1-60.raw", "Dab2-1-76.raw", "report.tsv", "checksum.txt", "Dab2-1-33.raw", "Dab2-1-16.raw", "Dab2-1-41.raw", "Dab2-1-68.raw", "Dab2-1-7.raw", "Dab2-1-50.raw", "Dab2-1-25.raw", "Dab2-1-8.raw", "Dab2-1-24.raw", "Dab2-1-42.raw", "Dab2-1-59.raw", "Dab2-1-40.raw", "Dab2-1-83.raw", "Dab2-1-23.raw", "Dab2-1-53.raw", "Dab2-1-66.raw", "Dab2-1-6.raw", "Dab2-1-10.raw", "Dab2-1-67.raw", "Dab2-1-49.raw", "Dab2-1-54.raw", "Dab2-1-36.raw", "Dab2-1-19.raw", "Dab2-1-84.raw", "Dab2-1-71.raw", "Dab2-1-52.raw", "Dab2-1-82.raw", "Dab2-1-22.raw", "Dab2-1-78.raw", "Dab2-1-79.raw", "Dab2-1-5.raw", "Dab2-1-65.raw", "Dab2-1-48.raw", "Dab2-1-18.raw", "FileLegend.xlsx", "Dab2-1-35.raw", "Dab2-1-70.raw", "Dab2-1-39.raw", "Dab2-1-64.raw", "Dab2-1-21.raw", "Dab2-1-4.raw", "Dab2-1-13.raw", "Dab2-1-12.raw", "Dab2-1-73.raw", "Dab2-1-80.raw", "Dab2-1-56.raw", "Dab2-1-47.raw", "Dab2-1-30.raw", "Dab2-1-11.raw", "Dab2-1-38.raw", "Dab2-1-63.raw", "Dab2-1-3.raw", "Dab2-1-29.raw", "Dab2-1-37.raw", "Dab2-1-55.raw", "Dab2-1-81.raw", "Dab2-1-72.raw", "Dab2-1-20.raw", "Dab2-1-46.raw", "Dab2-1-15.raw", "Dab2-1-58.raw", "Dab2-1-1.raw", "Dab2-1-75.raw", "Dab2-1-28.raw", "Dab2-1-2.raw", "Dab2-1-62.raw", "Dab2-1-32.raw", "Dab2-1-45.raw", "Dab2-1-27.raw", "Dab2-1-57.raw", "Dab2-1-14.raw", "Dab2-1-74.raw", "Dab2-1-61.raw", "Dab2-1-44.raw", "Dab2-1-31.raw" ], "highlights" : { } }, { "accession" : "PAD000033", "title" : "A lipid–immune network signature defines susceptibility to asparaginase-associated pancreatitis", "projectDescription" : "Asparaginase is essential for curing acute lymphoblastic leukemia (ALL), but its use is limited by asparaginase-associated pancreatitis (AAP), a severe and unpredictable toxicity lacking validated prospective biomarkers. To define early systemic features of susceptibility, we performed longitudinal lipidomic and proteomic profiling in two independent pediatric ALL cohorts (n = 159; 77 AAP cases, 82 controls) using paired blood samples collected before asparaginase exposure and by the end of ALL induction therapy (which included a single induction dose of asparaginase), thereby capturing pre-injury biology rather than consequences of pancreatitis. Across cohorts and analytic layers, we identify a reproducible lysophosphatidylcholine (LPC)–centered signature characterized by attenuated ALL induction therapy-associated LPC responses and disruption of LPC co-regulation at the network level. Proteomic profiling reveals concurrent enrichment of cytokine signaling pathways, and integrative analyses demonstrate altered lipid–cytokine coupling, including a flip in association direction for LPC species versus interleukin-18 (IL-18) between cases and controls. Although IL-18/LPC ratios do not differ globally, elevated post-induction IL-18/LPC ratios selectively identify AAP risk within a protocol-defined very high-risk ALL subgroup (AUC = 0.81). These findings support a systems-level model in which failure of coordinated lipid–immune responses under therapeutic stress confers vulnerability to AAP, providing a framework for prospective validation and network-informed mitigation strategies.", "dataProcessingProtocol" : "Initial, post-induction, and the ratio between the two timepoints for each protein were log2 transformed to better satisfy the normality assumption. Lipid species with a missing rate greater than 50% at each timepoint and in each group were excluded. Two-sample t-tests with unequal variance were performed to compare the lipid concentrations between cases and controls at each timepoint. For each sampling condition, differential abundance analysis was conducted using a linear mixed-effects model to account for lipids measured at two timepoints, adjusting for covariates to identify proteins that exhibited significant changes between cases and controls, and over time. Both P values and Q values were obtained using Benjamini–Hochberg method for FDR control.", "sampleProcessingProtocol" : "De-identified serum samples were collected from each subject recruited from Centre Hospitalier Universitaire Sainte-Justine, Montreal, enrolled in DFCI protocol 16-001 and frozen within one hour of collection. Serum samples from the validation cohort were submitted to Olink (Waltham, MA) for analysis with the Olink 3072 platform for proteomics analysis. The Olink platform utilizes proximity extension assay technology, enabling simultaneous quantification of multiple protein analytes with high sensitivity and specificity", "projectTags" : [ ], "keywords" : [ "Asparaginase associated pancreatitis", "Olink" ], "doi" : "10.6019/PAD000033", "submissionType" : "AFFINITY", "publicationDate" : "2026-03-31", "updatedDate" : "2026-03-25", "submissionDate" : "2026-03-25", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Cheng-Yu Tsai" ], "labPIs" : [ "Sohail Husain" ], "affiliations" : [ "Division of Pediatric Gastroenterology, Department of Pediatrics, Stanford University, Palo Alto, CA, 94304, USA." ], "instruments" : [ "Olink Explore 3072/384" ], "softwares" : [ ], "quantificationMethods" : [ "NPX" ], "sampleAttributes" : [ "Homo sapiens (Human)", "blood serum" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Blood serum" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Olink affinity proteomics", "Affinity proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "20211799_Tsai_NPX_2022-03-14.npx.csv" ], "highlights" : { } }, { "accession" : "PXD076043", "title" : "Cathelicidin derivative IDR-1018 exhibits anti-protease effects that alleviate Citrobacter rodentium-induced colitis", "projectDescription" : "Short, cationic, and amphipathic host defence peptides exhibit a wide range of anti-infective, anti-inflammatory, wound-healing, and antibiofilm activities. A synthetic peptide innate defence regulator (IDR) 1018, derived from the bovine neutrophil bactenecin, has been proposed as a therapeutic candidate for treating bacterial infections due to its immunomodulatory and antibacterial properties. IDR-1018 was examined as a treatment in murine colitis caused by the attaching/effacing enteropathogen, Citrobacter rodentium. IDR-1018, administered intra-peritoneally for three consecutive days after oral challenge with C. rodentium, mitigated microscopic lesions of colitis. Specifically, IDR-1018 reduced fecal protease levels, particularly elastase, by increasing the formation of inhibitory elastase–serpin complexes. IDR-1018 targeted neutrophils, the main effectors in colitis. Human and murine neutrophils stimulated and recruited by IDR-1018 exhibited reduced protease and elastase activity, as well as lower inflammatory cytokine production in response to inflammatory stimulation, showing functional reprogramming toward a less tissue-damaging phenotype. This study reveals a new activity of IDR-1018 in counteracting protease, which correspondingly attenuates C. rodentium colitis.", "dataProcessingProtocol" : "Spectra data obtained during mass-spectrometry were matched to peptide sequences from a mouse reference file obtained from Uniprot on March 20th, 2024, using DIA-NN (v.1.8.1). DIANN was run in “FASTA digest for library -free search mode” Variable modifications included N-term M excisions. Fixed modifications included Carbamidomethylation on Cystines. All other DIANN settings were set to default1. MSstatsShiny UI (v0.1.0; https://msstatsshiny.com/app/MSstatsShiny) running MSstats (v.4.2.0) was used to run the statistical analysis2. The DIANN file “Report.tsv” were used for the analysis, while the annotation file was created according to directions provided by MSstats. Unique peptides were included in the analysis. For data processing, log2 was defined for data transformation and equalize medians was the method chosen for normalization. The options that were enabled for this analysis were “Use unique peptides”, “remove proteins with 1 feature”, and “remove runs with over 50% missing values”. Statistical inference was performed using custom pairwise comparisons, and the data was imported to excel for final assessment. Significant enriched proteins for each group were found using a p-value cut-off of 0.05. Metascape analysis was used to identify enriched pathways3. Protein-protein interactions were encoded into networks using the metascape website (https://metascape.org/), and enriched pathways were plotted as heatmaps.", "sampleProcessingProtocol" : "The shotgun proteomic analysis was performed as described previously (10),. Briefly, tissue samples here bead bater homogenized. a small tissue samples was mixed with 500 ul of lysis buffer (1% SDS, 50 mM Amonium bicarbonate and protease inhibitor) with 3 stainless steal beads followzed by bead beating at 30 hz for 20 minutes using a Qiagen tissuelyser 2. Samples we centrifuged at 14 000 xg for 10 minutes to pellet unwanted debris. BCA quantification was performed on the supenatient according th manufacters directions (thermofisher) Samples were prepared using the filter-assisted separation of peptides (FASP) method4. 100 ug of protein was precipitated by adding trichloroacetic acid (TCA, 25% v/v final) followed by an incubation on ice. Samples were then centrifuged at 14,000 xg for 15 minutes at 4°C, washed 3 times in ice cold acetone, and stored at -20°C overnight. Samples were resuspended in 8M tris-urea solution by shaking and then denatured with the addition of 10 mM DTT at 37°C for 30 minutes. Samples were moved to the top of a 30 kDa filter and were then centrifuged at 14,000 x g for 15 minutes. 50 mM iodoacetamide was added in the dark at room temperature to complete carbamidomethyl modification of the cystines. Samples were then washed 3 times with 100 ul of 8 M tris-urea and 3 times with followed by 3 washes in 100 ul of 50 mM ammonium bicarbonate. The samples were trypsinized at 37°C overnight at a ratio of 1:10 trypsin: total protein, and subsequently, eluted off the filter membrane by washing two times with 50 mM ammonium bicarbonate. Finally, samples were subjected to a c18 clean up with waters solid-phase extraction (SPE) column, according to directions from the manufacturer. High performance liquid chromatography (HPLC) and mass spectrometry (MS) Tryptic peptides were analyzed on an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific) operated with Xcalibur (version 4.4.16.14) and coupled to a Thermo Scientific Easy-nLC (nanoflow Liquid Chromatography) 1200 system. A total mass of 2 μg tryptic peptides were loaded onto a C18 trap (75 um x 2 cm; Acclaim PepMap 100, P/N 164946; ThermoScientific) at a flow rate of 2ul/min of solvent A (0.1% formic acid in LC-MS grade water). Peptides were eluted using a 120 min gradient from 5 to 40% (5% to 28% in 105 min followed by an increase to 40% B in 15 min) of solvent B (0.1% formic acid in 80% LC-MS grade acetonitrile) at a flow rate of 0.3 μL/min and separated on a C18 analytical column (75 um x 50 cm; PepMap RSLC C18; P/N ES803; ThermoScientific). The mass spec was opetated in DIA mode using the default settings from the manufacturer. Peptides were then electrosprayed using 2.1 kV voltage into the ion transfer tube (300°C) of the Orbitrap Lumos operating in positive mode. The Orbitrap first performed a full MS scan at a resolution of 120000 FWHM to detect the precursor ion having a m/z between 380 and 985 and a +2 to +7 charge. The Orbitrap AGC (Auto Gain Control) and the maximum injection time were set at standard and 50 ms, respectively. The Orbitrap was operated using the top speed mode with a 3 sec cycle time for precursor selection. The fragment ions (MS2) were analyzed in the orbitrap with a 40 sliding windows of 16.0072 m/z starting at 379.4224. The precurson ions within the sliding windows were sleected for MS2 with HCD (30% collision energy) in the ion routing multipole. The AGC and the maximum injection time were set at 1e4 and 35 ms, respectively, for the quadrupole.", "projectTags" : [ ], "keywords" : [ "Host defence peptides; colitis; citrobacter rodentium." ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-14", "updatedDate" : "2026-03-24", "submissionDate" : "2026-03-24", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Daniel Young" ], "labPIs" : [ "Antoine Dufour" ], "affiliations" : [ "Cumming School of Medicine, Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, T2N 4N1, Canada." ], "instruments" : [ "Orbitrap Fusion Lumos" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "colon", "Mus musculus (Mouse)" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Colon" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-independent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "S20002_217_Cobo_CR_Peptide_16_260301.raw", "S19990_205_Cobo_CR_Peptide_4_260228.raw", "S19991_206_Cobo_CR_Peptide_5_260228.raw", "S19993_208_Cobo_CR_Peptide_7_260228.raw", "S20000_215_Cobo_CR_Peptide_14_260301.raw", "S19998_213_Cobo_CR_Peptide_12_260228.raw", "S19992_207_Cobo_CR_Peptide_6_260228.raw", "S19996_211_Cobo_CR_Peptide_10_260228.raw", "checksum.txt", "S19987_202_Cobo_CR_Peptide_1_260228.raw", "2026-3-6_Cobo_CR_peptide.zip", "S19989_204_Cobo_CR_Peptide_3_260228.raw", "S19994_209_Cobo_CR_Peptide_8_260228.raw", "S19995_210_Cobo_CR_Peptide_9_260228.raw", "S20001_216_Cobo_CR_Peptide_15_260301.raw", "S19999_214_Cobo_CR_Peptide_13_260301.raw", "S19997_212_Cobo_CR_Peptide_11_260228.raw", "S19988_203_Cobo_CR_Peptide_2_260228.raw" ], "highlights" : { } }, { "accession" : "PXD076080", "title" : "Clinical and Proteomic Predictors of Primary Response to Infliximab in Crohn’s Disease with Small Bowel Involvement: A Pilot Study in China", "projectDescription" : "Background and Aim: Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract. Although various biomarkers have been used to predict infliximab (IFX) response in CD, its efficacy in patients with small bowel involvement remains underexplored. This study combined clinical data with serum proteomics to develop prediction models for IFX efficacy at week 14 in small bowel-involved CD. Methods: In this pilot study, 41 patients with small bowel-involved CD treated with IFX between January 2020 and December 2023 at two Chinese hospitals were included. Clinical response and remission at week 14 were assessed using the Crohn’s Disease Activity Index (CDAI). Pre-treatment serum proteomics identified differentially expressed proteins (fold change >1.2, p < 0.05). Predictive models were developed via logistic regression and validated with ROC curve analysis using SPSS 24.0 and R 4.4.0 (p < 0.05). Results: Clinical analysis identified extraintestinal manifestations (EIM), hemoglobin <87.5 g/L, and a history of CD-related surgery as key predictors of primary non-response (AUC = 0.846, 95% CI: 0.715–0.978). Proteomic analysis revealed 13 differentially expressed proteins (individual AUCs: 0.588-0.868). A serum model combining MSN, SAA4, VNN1, and IGFBP5 achieved an AUC of 0.931 (95% CI: 0.845-1.000). Integrating MSN with clinical predictors yielded combined models with AUCs between 0.882 and 0.914; notably, the MSN + CD-related surgery model performed best (AUC = 0.914, 95% CI: 0.829–0.998). Conclusions: The developed clinical, serum, and combined prediction models effectively forecast IFX efficacy in small bowel-involved CD, providing valuable tools for personalized treatment strategies.", "dataProcessingProtocol" : "Raw MS data were processed using MaxQuant software (version not specified) against the human Swiss-Prot database downloaded from UniProt. The search parameters were set as follows: precursor intensity fraction (PIF) filter at 0.75, reporter ion mass tolerance of 0.003 Da, carbamidomethylation of cysteine as a fixed modification, and trypsin as the protease with a maximum of two missed cleavages. The initial precursor mass tolerance was set to 20 ppm for the first search and 4.5 ppm for the main search. The minimum peptide length was set to seven amino acids. False discovery rates (FDR) at the peptide, protein, and site levels were all set to 1%.", "sampleProcessingProtocol" : "The depletion column was equilibrated to room temperature for 10–15 minutes, and the resin was gently resuspended by inversion. A 10 μL aliquot of serum was applied to the column, which was then inverted several times to ensure complete mixing. The column was rotated at room temperature for 1 hour, followed by centrifugation at 1,000 × g for 2 minutes in a 2 mL collection tube. The flow-through containing the depleted serum (14 high-abundance proteins removed) was retained. A 200 μL aliquot of the depleted sample was mixed with 1 mL of pre-chilled acetone, vortexed, and incubated at –20 °C for 1 hour. Following centrifugation at 15,000 × g for 20 minutes at 4 °C, the supernatant was discarded, and the protein pellet was collected. Protein digestion and desalting were performed using a pretreatment kit (OSFP0001 8X, Yiwei Micro). The protein pellet was reconstituted in 20 μL of Reagent A with thorough mixing, followed by the addition of 4 μL of Reagent B. The mixture was heated at 95 °C for 5 minutes, cooled to room temperature, and 15 μL of Reagent C was added. Enzymatic digestion was carried out at 37 °C for 6 hours, terminated by adding 3 μL of Reagent D. The resulting precipitate was removed by centrifugation at 15,000 × g for 10 minutes, and the supernatant was subjected to desalting. Peptide desalting was performed using a spin column. The column was sequentially activated with 100 μL methanol, conditioned with 100 μL Condition Buffer (70% acetonitrile, 0.2% trifluoroacetic acid [TFA]), and equilibrated with 100 μL Wash Buffer (0.2% TFA), each step followed by centrifugation at 700 × g for 1 minute. The sample pH was adjusted to below 3 prior to loading onto the column, which was centrifuged at 700 × g for 1 minute; this loading step was repeated once. The column was washed twice with 100 μL Wash Buffer (700 × g, 1 minute each), and peptides were eluted twice with 50 μL Elution Buffer (90% acetonitrile, 0.2% TFA) (700 × g, 1 minute each), yielding a final volume of 100 μL. The eluted peptides were concentrated using a vacuum centrifuge and stored at –80 °C until analysis. For nano-HPLC-MS/MS analysis, the peptides were reconstituted in solvent A (0.1% formic acid in water) and separated on an Easy nLC 1000 UHPLC system (Thermo Fisher Scientific) coupled to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). Peptides were loaded onto a C18 analytical column (Thermo C18, 75 μm × 25 cm) and separated at a flow rate of 300 nL/min with a column temperature of 55 °C using a 60-minute gradient: 4% solvent B (0.1% formic acid in acetonitrile) from 0 to 5 min, 4–11% B from 5 to 8 min, 11–25% B from 8 to 47 min, 25–60% B from 47 to 52 min, 60–80% B from 52 to 54 min, 80% B from 54 to 56 min, 80–4% B from 56 to 58 min, and 4% B from 58 to 60 min. The mass spectrometer was operated in positive ion mode with a spray voltage of 1,800 V and an ion transfer tube temperature of 320 °C. Data were acquired in data-dependent acquisition (DDA) mode using Xcalibur software. Full-scan MS spectra were acquired at a resolution of 120,000 at m/z 200 over a mass range of 350–1,700 m/z with an automatic gain control (AGC) target of 3e6 and a maximum injection time of 50 ms. The top 20 most intense precursor ions were selected for fragmentation with a resolution of 60,000 at m/z 200, a normalized collision energy (NCE) of 32%, an isolation window of 1.0 m/z, an AGC target of 1e5, a maximum injection time of 120 ms, and a dynamic exclusion duration of 40 seconds. Precursors with charge states 2–6 were selected for MS/MS, with fragmentation starting at m/z 105.", "projectTags" : [ ], "keywords" : [ "Small bowel involvement", "Infliximab", "Prediction model", "Proteomics", "Primary non-response", "Crohn’s disease" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-26", "updatedDate" : "2026-03-24", "submissionDate" : "2026-03-24", "downloadCount" : 9, "avgDownloadsPerFile" : 0.20930232, "botCount" : 4, "hubCount" : 4, "organicCount" : 1, "percentile" : 1, "yearlyDownloads" : [ { "year" : "2026", "count" : 9 } ], "submitters" : [ "Yaqing Bai" ], "labPIs" : [ "Xiaoyin Bai" ], "affiliations" : [ "No.1 Shuaifuyuan Wangfujing Dongcheng District, Beijing, China 100730" ], "instruments" : [ "Q Exactive HF" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "blood serum" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Blood serum" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "63.msf", "38.msf", "68.msf", "25.msf", "50.msf", "64.msf", "34.msf", "69.msf", "04.msf", "67.msf", "checksum.txt", "73.msf", "11.msf", "29.msf", "51.msf", "03.msf", "16.msf", "72.msf", "20.msf", "33.msf", "07.msf", "36.msf", "14.msf", "66.msf", "15.msf", "58.msf", "75.msf", "32.msf", "43.msf", "18.msf", "62.msf", "search.xlsx", "13.msf", "65.msf", "39.msf", "40.msf", "27.msf", "09.msf", "57.msf", "22.msf", "35.msf", "05.msf", "70.msf" ], "highlights" : { } }, { "accession" : "PXD076071", "title" : "Cell size-dependent mRNA transcription drives proteome remodeling", "projectDescription" : "Increasing cell size drives proteomic changes that impact cell physiology. However, the molecular basis of size-dependent proteome remodeling has remained unclear. Here, we develop an inducible Cyclin D1 expression system in human cells to generate populations of proliferating cells spanning over a two-fold size range. We use this genetic system to make comprehensive genome-wide measurements of mRNA and protein concentrations and stability. We find that protein and mRNA turnover rates are weakly related to cell size, but that mRNA concentrations are strongly size-dependent. This establishes that transcriptional regulation is the basis of proteome remodeling. Live-cell imaging of endogenous mRNAs using MS2 fluorescent protein binding motifs is used to measure how transcriptional dynamics change with cell size. Larger cells prolong transcriptional bursts and shorten inactive periods between bursts but maintain similar burst amplitudes to achieve transcriptional scaling. Taken together, our results show how transcription is modulated by cell size to remodel the proteome and alter cell physiology.", "dataProcessingProtocol" : "All raw files were searched using the Andromeda engine embedded in MaxQuant (v2). Reporter ion MS3 search was conducted using TMT10-plex settings. Variable modifications were oxidation (M) and protein N-terminal acetylation. For pulse SILAC experiments, carbamidomethyl (C) was a fixed modification. The number of modifications per peptide was capped at five. Digestion was tryptic (proline-blocked). Database search used the UniProt Human proteome. The minimum peptide length was 7 amino acids. 1% FDR was determined using a reverse decoy proteome. Peptides were first filtered out for decoy and contaminants. Each TMT channel was normalized by the total intensity in all TMT channels that were loaded together. Any peptides with the resulting ratio values 0 or less were filtered out. Each peptide intensity was normalized by the mean of all peptide intensities in each TMT channel. This value was used as a measure of relative concentration of the peptide in the given TMT channel. To get the change in relative concentration of the peptide with cell size, we transformed the concentration values of the peptide and the respective originating cell’s volumetric mean-normalized measurements by log2. We then drew a linear regression line using np.polyfit and obtained the slope value for that peptide. To find the change in concentration of a protein with cell size increase, the slope values for all peptides matching to that protein were averaged. Only proteins with at least two unique peptide matches were carried forward for further analysis. Peptides were differentiated by their modified sequence, charge, fraction of loading, and leading razor protein.", "sampleProcessingProtocol" : "cells were trypsinized and pelleted by centrifugation at 1000xG for 5 minutes and lysed for 30 minutes on ice in RIPA lysis buffer (Abcam; ab156034) with a protease and phosphatase inhibitor cocktail (Thermo Scientific; 78440). Lysates were cleared by centrifugation at 16000xG for 10 minutes at 4℃. Samples were then denatured by 1% SDS, reduced with 5mM DTT, alkylated with 10mM iodoacetamide, then precipitated with three volumes of 50% acetone and 50% ethanol. Proteins were solubilized with 2M urea, 50 mM Tris-HCl, pH 8.0, and 150mM NaCl, then digested with TPCK-treated trypsin (50:1) overnight at 37℃. Following digestion, peptides were acidified with trifluoroacetic acid and desalted with Sep-Pak 50mg C18 columns (Sep-Pak; WAT054955). The columns were pre-conditioned with 80% acetonitrile and 0.1% acetic acid, and washed with 0.1% trifluoroacetic acid. After loading the peptides, the columns were then washed with 0.1% acetic acid and eluted with 80% acetonitrile and 0.1% acetic acid. The eluents were dried in a concentrator at 45℃.", "projectTags" : [ ], "keywords" : [ "Transcription", "Size scaling", "Cell size", "Protein turnover" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-03", "updatedDate" : "2026-03-24", "submissionDate" : "2026-03-24", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Michael Lanz" ], "labPIs" : [ "Jan Skotheim" ], "affiliations" : [ "Department of Biology, Stanford University" ], "instruments" : [ "Orbitrap Fusion Lumos" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "epithelial cell" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Epithelial cell" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "msms_G1_G2_2.txt", "FL0032522.raw", "FL2002603.raw", "FL2003000.raw", "FL2001671.raw", "FL2003001.raw", "FL2002981.raw", "FL2002611.raw", "FL2002972.raw", "FL2002998.raw", "checksum.txt", "FL2001672.raw", "FL2002990.raw", "FL2001673.raw", "FL2003002.raw", "G1vsG2_FACSsize_F0_R1_Tmulti_DDATMTMS3001_000356_JASK006.raw", "FL2002973.raw", "FL2002999.raw", "FL2002982.raw", "FL2002978_20221105122002.raw", "FL2002988.raw", "FL0032520.raw", "FL2002975.raw", "FL0032195.raw", "FL2002992.raw", "FL0034282.raw", "FL2002974.raw", "FL2002987.raw", "FL2002613.raw", "FL2002609.raw", "FL2001670.raw", "FL2002989.raw", "msms_G1_G2_1.txt", "FL2002993.raw", "FL2002980.raw", "FL2001666.raw", "FL2002969.raw", "FL2002994.raw", "msms_turnover.txt", "FL2002977.raw", "FL2002968.raw", "FL0032526.raw", "FL2001667.raw", "FL2002995.raw", "FL2001669.raw", "FL0034278.raw", "FL2002607.raw", "FL2002986.raw", "FL2001668.raw", "FL2002983.raw", "FL2002605.raw", "FL2002979.raw", "FL0032524.raw", "FL2002996.raw", "msms_cell_size_system.txt", "FL2002970.raw", "FL2002971.raw", "G1vsG2_FACSsize_F0_R1_Tmulti_DDATMTMS3001_000356_JASK006_20231031123122.raw", "FL2002997.raw", "FL2002984.raw" ], "highlights" : { } }, { "accession" : "PXD075984", "title" : "Dengue virus recombinant NS1 sample LC-MSMS", "projectDescription" : "LC-MS/MS results of dengue virus serotype 2 recombinant NS1 co-purified samples", "dataProcessingProtocol" : "Raw data were processed using PEAKS Studio (version 13.0). Spectra were searched against the SwissProt Homo sapiens database (release 2025_03), a universal contaminants database, and the flavivirus polyprotein sequence database. Search parameters included a precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.02 Da, allowing up to two missed cleavages. Variable modifications included N-terminal acetylation, methionine oxidation, phosphorylation (STY), and carbamidomethylation. Protein identifications were filtered at a 1% false discovery rate (FDR) at both peptide-spectrum match and protein levels, requiring at least one unique peptide per protein.", "sampleProcessingProtocol" : "Recombinant secreted NS1 (sNS1) from DENV2 strain PVP94/07 and sNS1-mVenus were expressed in Expi293F cells following transient transfection. Culture supernatants were harvested 4–5 days post-transfection, clarified by centrifugation and filtration, and purified by Ni-affinity chromatography. Eluted proteins were buffer exchanged and further purified by anion exchange chromatography (Resource Q) followed by size-exclusion chromatography (Superdex 200 Increase 10/300 GL). Protein purity was assessed by SDS–PAGE. For proteomic analysis, purified protein samples were lysed in S-TRAP lysis buffer (50 mM triethylammonium bicarbonate, 5% SDS) and processed using S-TRAP Micro columns according to the manufacturer’s protocol. Peptides were separated using an EvoSep One LC system equipped with an Ionopticks Aurora Elite XT C18 column (75 μm × 150 mm, 1.7 μm particles) using the Whisper Zoom 40 SPD method. Mobile phases consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Mass spectrometry analysis was performed on a Thermo Fisher Orbitrap Exploris 480 operating in positive ion mode with a spray voltage of 1.5 kV. Data-dependent acquisition (DDA) was used with a full scan range of 400–1600 m/z at 60,000 resolution followed by MS/MS scans at 15,000 resolution with HCD fragmentation (normalized collision energy 30%).", "projectTags" : [ ], "keywords" : [ "Denv", "Ns1", "Recombinant" ], "doi" : "10.6019/PXD075984", "submissionType" : "COMPLETE", "publicationDate" : "2026-03-25", "updatedDate" : "2026-03-22", "submissionDate" : "2026-03-22", "downloadCount" : 3, "avgDownloadsPerFile" : 0.33333334, "botCount" : 1, "hubCount" : 2, "organicCount" : 0, "percentile" : 0, "yearlyDownloads" : [ { "year" : "2026", "count" : 3 } ], "submitters" : [ "Qunfei Zhou" ], "labPIs" : [ "Shee-Mei Lok" ], "affiliations" : [ "Duke-NUS Medical School, Singapore" ], "instruments" : [ "LTQ Orbitrap" ], "softwares" : [ ], "quantificationMethods" : [ "Spectrum counting", "Peptide counting", "Protein Abundance Index - PAI" ], "sampleAttributes" : [ "Homo sapiens (Human)", "early embryonic cell" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Early embryonic cell" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition", "Top-down proteomics" ], "sdrf" : "", "projectFileNames" : [ "251105_2510S15_1_raw.mgf", "checksum.txt", "251105_2510S15_1_mzidentml.xml", "251105_2510S15_1.raw", "251105_2510S15_2_raw.mgf", "251105_2510S15_2_mzidentml.xml", "251105_2510S15_1.mgf", "251105_2510S15_2.mgf", "251105_2510S15_2.raw" ], "highlights" : { } }, { "accession" : "PXD075982", "title" : "TurboID-based proximity labeling of ER-localized TMEM120A/B interactome in Oleic Acid-treated HEK293T cells", "projectDescription" : "Raw mass spectrometry data (.d raw data from Bruker) supporting the study: \"A conserved ER protein prevents lipotoxicity by stimulating the key enzyme in glycerolipid synthesis\". This dataset contains LC-MS/MS raw files from a proximity labeling experiment (TurboID) designed to identify interacting proteins of ER-localized TMEM120A/B during lipid droplet biogenesis.", "dataProcessingProtocol" : "Raw mass spectrometry data (.d raw data from Bruker) supporting the study: \"A conserved ER protein prevents lipotoxicity by stimulating the key enzyme in glycerolipid synthesis\". This dataset contains LC-MS/MS raw files from a proximity labeling experiment (TurboID) designed to identify interacting proteins of ER-localized TMEM120A/B during lipid droplet biogenesis. Cell Model: HEK293T cells expressing either TurboID-TMEM120A/B (experimental) or TurboID-NES (negative control). Treatment: All samples were treated with Oleic Acid (OA) to induce lipid droplet formation.", "sampleProcessingProtocol" : "Cell Model: HEK293T cells expressing either TurboID-TMEM120A/B (experimental) or TurboID-NES (negative control). Treatment: All samples were treated with Oleic Acid (OA) to induce lipid droplet formation. Sample Type: Streptavidin-enriched ER membrane fractions. Detailed experimental procedures, including biotinylation, cell fractionation, and data analysis parameters, are described in the associated manuscript.", "projectTags" : [ ], "keywords" : [ "Lipid droplet", "Tag", "Turboid", "Proximity labeling" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-09", "updatedDate" : "2026-03-22", "submissionDate" : "2026-03-22", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Siwei Huang" ], "labPIs" : [ "Ho Yi Mak" ], "affiliations" : [ "The Hong Kong University of Science and Technology" ], "instruments" : [ "maXis" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "epithelial cell" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Epithelial cell" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "diaPASEF", "Data-independent acquisition" ], "sdrf" : "", "projectFileNames" : [ "report.gg_matrix.tsv", "report-first-pass.gg_matrix.tsv", "report.pr_matrix.tsv", "report-first-pass.pr_matrix.tsv", "report.stats.tsv", "TB_A2_Slot2-8_1_1965.mzML", "TB_B2_Slot2-17_1_1968.mzML", "TB_B1_Slot2-16_1_1967.mzML", "report-first-pass.tsv", "report-first-pass.stats.tsv", "report.tsv", "checksum.txt", "report.pg_matrix.tsv", "report-first-pass.pg_matrix.tsv", "TB_A1_Slot2-7_1_1964.mzML", "TB_N3_Slot2-27_1_1972.mzML", "TB_A3_Slot2-9_1_1966.mzML", "TB_B3_Slot2-18_1_1969.mzML", "report.unique_genes_matrix.tsv", "sdrf_form.csv", "TB_N1_Slot2-25_1_1970.mzML", "report-first-pass.unique_genes_matrix.tsv", "TB_N2_Slot2-26_1_1971.mzML" ], "highlights" : { } }, { "accession" : "PXD075986", "title" : "Activated T cell extracellular vesicle DNA transfer enhances antigen presentation and anti-tumor immunity", "projectDescription" : "Antigen processing and presentation (APP) is essential for adaptive immunosurveillance. We uncover a mechanism whereby activated T cell-derived extracellular vesicles (AT EVs ) drive a positive feedback loop that enhances antigen presentation and immune responses in normal physiology and cancer. AT EV -induced immunogenicity relies on extracellular vesicular double-stranded DNA (EV DNA ), which is notably abundant and primarily composed of genomic DNA enriched in immune-related genes, including those encoding APP machinery. Mechanistically, granzyme B (Gzmb) packaged by AT EVs disrupts the nuclear envelope of recipient cells, facilitating intranuclear transfer and subsequent transient expression of EV DNA encoding APP genes. DNase treatment removes most AT-EV DNA , abrogating APP upregulation and thus T cell activation and recruitment to tumors. Notably, AT EVs hold promise as an acellular immunotherapy, restoring APP and synergizing with checkpoint blockade in immunotherapy-refractory tumors. Collectively, our findings uncover a mechanism of transient, non-viral gene delivery by AT EVs which boosts APP and anti- tumor immunity while limiting autoimmunity.", "dataProcessingProtocol" : "Generated LC-MS/MS data was processed using Proteome Discoverer/Mascot v2.5. Mouse or human data was queried against UniProt’s mouse or human cell culture database concatenated with Trypsin/Lys-C sequences. Identified proteins without contaminants were utilized for subsequent analysis. Abundance (average area of the 3 most intense peptides for a protein group) of identified proteins were evaluated by principal component analysis (PCA) using proteiNorm 81 with default settings for consistency in the replicates. Fold changes (FCs) and significance of protein abundance were calculated by using raw data log 2 transformation and normalization, followed by missing data imputation, F-test, log 2 FC, and P-value determination. Heatmaps were generated based on normalized abundance of identified proteins using the online analysis tool Heatmapper and Excel.", "sampleProcessingProtocol" : "EV samples were prepared for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis at the Rockefeller University Proteomics Resource Center as previously described. Briefly, 5 µg of EV samples were dried by vacuum centrifugation and re-dissolved in 30-50µL 8M urea/50mM ammonium bicarbonate/10mm DTT. Following lysis and reduction, proteins were alkylated using 20 or 30mM iodoacetamide (Sigma-Aldrich). Proteins were digested with Endopeptidase Lys-C (Wako) in less than 4M urea followed by trypsination (Promega) in less than 2M urea. Peptides were desalted and concentrated using Empore C18-based solid phase extraction prior to analysis by high resolution/high mass accuracy reversed phase (C18) nano-LC-MS/MS. Peptides were separated on a C18 column (12 cm / 75 mm, 3 mm beads, Nikkyo Technologies) at 200 or 300 nl min -1 with a gradient increasing from 1% Buffer B/95% buffer A to 40% buffer B/60% Buffer A in typically 90 or 120 min (buffer A: 0.1% formic acid, buffer B: 0.1% formic acid in 80% acetonitrile). Mass spectrometers (Q-Exactive-HF or LUMOS) were operated in data dependent positive ion mode.", "projectTags" : [ ], "keywords" : [ "T-cell", "Dna transfer", "Extracellular vesicle" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-23", "updatedDate" : "2026-03-22", "submissionDate" : "2026-03-22", "downloadCount" : 60, "avgDownloadsPerFile" : 4.0, "botCount" : 45, "hubCount" : 11, "organicCount" : 4, "percentile" : 3, "yearlyDownloads" : [ { "year" : "2026", "count" : 60 } ], "submitters" : [ "henrik molina" ], "labPIs" : [ "David Lyden" ], "affiliations" : [ "1 Children’s Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children’s Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA" ], "instruments" : [ "Orbitrap Fusion Lumos", "Q Exactive HF" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "cell culture", "Homo sapiens (Human)" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Cell culture" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "MS238258LUM_Mengying_Lyden_hATexo1.raw", "MS227362QEHF_Mengying_Lyden_Activated_T-2.raw", "MS238258LUM_Mengying_Lyden_hATexo3.raw", "MS238258LUM_Mengying_Lyden.xlsx", "MS238258LUM_Mengying_Lyden_hNTexo3.raw", "MS227362QEHF_Mengying_Lyden_Activated_T-1.raw", "MS227362QEHF_Mengying_Lyden_naive_T-1.raw", "MS238258LUM_Mengying_Lyden_hNTexo4.raw", "checksum.txt", "MS227362QEHF_Mengying_Lyden_naive_T-2.raw", "MS238258LUM_Mengying_Lyden_hATexo2.raw", "MS238258LUM_Mengying_Lyden_hNTexo2.raw", "MS238258LUM_Mengying_Lyden_hNTexo1.raw", "MS238258LUM_Mengying_Lyden_hATexo4.raw", "MS227362_Mengying_Lyden.xlsx" ], "highlights" : { } }, { "accession" : "PXD075910", "title" : "IP-MS analysis of RLF/ZFP292-FLAG", "projectDescription" : "We performed immunoprecipitation followed by LC-MS/MS (IP-MS) in mouse embryonic stem cells in which endogenous RLF or ZFP292 was tagged with FLAG. This approach enabled the identification of proteins associated with RLF/ZFP292. The dataset provides a resource to investigate the composition of RLF/ZFP292-associated chromatin regulatory complexes.", "dataProcessingProtocol" : "Raw MS data were processed and searched against the mouse proteome database (UniProtKB, released January 2025) using Mascot (v2.3.2) for LXQ data.", "sampleProcessingProtocol" : "Approximately 3 × 10^7 cells were cultured in in three 15-cm dishes. Cells were collected with 0.1 M NaCl CSK buffer (10 mM PIPES, pH 7.0; 100 mM NaCl; 300 mM sucrose; 1 mM MgCl2; 1 mM EGTA; 0.1% Triton X-100). Permeabilized cells were treated with 25 U/ml KANEKA endonuclease (KANEKA, KEN02100) at 4°C for 30 min to digest all nucleic acids. The NaCl concentration was then increased to 0.5 M to stop the digestion and solubilize proteins. The lysate was centrifuged at 16,000 × g for 20 min, and the supernatant was used as input for immunoprecipitation. The input was incubated with anti-FLAG M2 affinity gel beads (Millipore, A2220) at 4°C for 2 h. Beads were washed with 0.5 M NaCl CSK buffer, and bound proteins were eluted using 3× FLAG peptide (F4799, Millipore). Eluted samples were separated by SDS-PAGE and cut into nine gel slices, followed by in-gel trypsin digestion. LC-MS/MS was performed using reverse-phase chromatography coupled to an LXQ Linear Ion Trap mass spectrometer (Thermo Fisher Scientific)", "projectTags" : [ ], "keywords" : [ "Zfp292", "Ip-ms", "Mescs", "Rlf" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-21", "updatedDate" : "2026-03-20", "submissionDate" : "2026-03-20", "downloadCount" : 35, "avgDownloadsPerFile" : 0.6363636, "botCount" : 3, "hubCount" : 32, "organicCount" : 0, "percentile" : 2, "yearlyDownloads" : [ { "year" : "2026", "count" : 35 } ], "submitters" : [ "Takamasa Ito" ], "labPIs" : [ "Chikashi Obuse" ], "affiliations" : [ "Laboratory of Genome Structure and Function Department of Biological Sciences Graduate School of Science The University of Osaka" ], "instruments" : [ "LXQ" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "embryonic stem cell", "cell culture", "Mus musculus (Mouse)" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Cell culture", "Embryonic stem cell" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "F066851.dat", "F066869.dat", "F066860.dat", "b_FLAG_Rlf_221111_221115_07.RAW", "c_FLAG_Zfp292_221111_221115_04.RAW", "a_TT2_WT_221111_221115_05.RAW", "a_TT2_WT_221111_221115_03.RAW", "checksum.txt", "F066868.dat", "c_FLAG_Zfp292_221111_221115_03.RAW", "F066850.dat", "b_FLAG_Rlf_221111_221115_08.RAW", "a_TT2_WT_221111_221115_04.RAW", "F066859.dat", "b_FLAG_Rlf_221111_221115_04.RAW", "F066867.dat", "F066854.dat", "a_TT2_WT_221111_221115_01.RAW", "c_FLAG_Zfp292_221111_221115_06.RAW", "F066849.dat", "F066852.dat", "b_FLAG_Rlf_221111_221115_05.RAW", "F066865.dat", "F066866.dat", "c_FLAG_Zfp292_221111_221115_05.RAW", "F066848.dat", "b_FLAG_Rlf_221111_221115_06.RAW", "F066853.dat", "a_TT2_WT_221111_221115_02.RAW", "F066870.dat", "F066856.dat", "a_TT2_WT_221111_221115_09.RAW", "b_FLAG_Rlf_221111_221115_02.RAW", "F066864.dat", "F066847.dat", "F066863.dat", "c_FLAG_Zfp292_221111_221115_08.RAW", "a_TT2_WT_221111_221115_08.RAW", "b_FLAG_Rlf_221111_221115_03.RAW", "F066855.dat", "F066846.dat", "c_FLAG_Zfp292_221111_221115_07.RAW", "b_FLAG_Rlf_221111_221115_09.RAW", "c_FLAG_Zfp292_221111_221115_02.RAW", "F066862.dat", "F066858.dat", "a_TT2_WT_221111_221115_07.RAW", "F066845.dat", "F066857.dat", "c_FLAG_Zfp292_221111_221115_01.RAW", "a_TT2_WT_221111_221115_06.RAW", "b_FLAG_Rlf_221111_221115_01.RAW", "F066861.dat", "c_FLAG_Zfp292_221111_221115_09.RAW", "F066844.dat" ], "highlights" : { } }, { "accession" : "PXD075951", "title" : "Sucrose-Activated TOR and phyA Signaling Alleviates Shade-Mediated Inhibition of Leaf Development in Arabidopsis", "projectDescription" : "To investigate how the sucrose-TOR signaling pathway regulates phyA, we conducted LC-MS/MS analysis to identify phosphorylation sites on phyA. In vivo, the phyA protein was immunoprecipitated from 35S::PHYA-YFP/phyA-211 seedlings treated with shade and 30 mM sucrose for 6 hours. In vitro, we used recombinant phyA protein purified from insect cells as the substrate and endogenous TOR complex immunoprecipitated from the 35S::LST8-1-FLAG transgenic line as the kinase source. Phosphopeptide enrichment and LC–MS/MS analysis of the in vitro reaction were performed.", "dataProcessingProtocol" : "Raw data were processed against the Arabidopsis TAIR database (32,790 entries) using Proteome Discoverer (version 2.4, Thermo Fisher Scientific) with the Mascot search engine (version 2.7.0, Matrix Science). The precursor and fragment mass tolerances were set to 10 ppm and 0.05 Da, respectively. Up to two missed cleavages were allowed. Carbamidomethylation of cysteine was set as a fixed modification, while oxidation of methionine and protein N-terminal acetylation were set as variable modifications. Phosphorylation of serine, threonine, and tyrosine residues was included as a variable modification for phosphopeptide analysis.", "sampleProcessingProtocol" : "Peptide digestion was performed using the FASP protocol with Microcon PL-10 filters. After three rounds of buffer exchange with 8 M urea in 100 mM Tris-HCl (pH 8.0), proteins were reduced with 10 mM DTT at 37 °C for 30 min and alkylated with 30 mM iodoacetamide at 25 °C for 45 min in the dark. Samples were then exchanged into digestion buffer (30 mM Tris-HCl, pH 8.0) and digested with trypsin (enzyme-to-protein ratio 1:50) at 37 °C for 12 h. The filtrates were collected, combined after washing the filter twice with 15% ACN, and vacuum-dried. Phosphopeptides were enriched using home-made TiO2 microcolumns (peptide: TiO2 = 1:10). Peptides were dissolved in loading buffer (1 M glycolic acid, 80% ACN, 5% TFA), loaded twice, washed with loading buffer followed by washing buffer (80% ACN, 1% TFA), and sequentially eluted with 2 M ammonium hydroxide and 1 M ammonium hydroxide in 30% ACN. Eluates were vacuum-dried. For the In vivo assay, LC–MS analysis was performed using an EASY-nLC 1200 system (Thermo Fisher Scientific) coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a home-made C18 analytical column (75 μm inner diameter × 25 cm, 1.9 μm) using a one-column configuration. The mobile phases consisted of solution A (0.1% formic acid) and solution B (0.1% formic acid in 80% ACN). Peptides were eluted at a flow rate of 200 nL min-1 using a gradient of 5–8% B (2 min), 8–44% B (38 min), 44–70% B (8 min), 70–100% B (2 min), followed by 100% B (10 min). FAIMS was enabled with compensation voltages of −40 and −60 V. MS1 spectra were acquired at a resolution of 60,000, and precursor ions with charge states of 2–7 were selected for MS2 analysis. A dynamic exclusion of 45 s was applied with a cycle time of 1 s. MS2 spectra were acquired with HCD fragmentation (isolation window 1.6 m/z; resolution 15,000; normalized collision energy 30%; maximum injection time 30 ms). For the In vitro assay, LC–MS analysis was performed using a nanoElute system (Bruker Daltonics) coupled to a timsTOF HT mass spectrometer (Bruker Daltonics) operating in PASEF mode. Peptides were separated on a C18 analytical column (75 μm inner diameter × 25 cm, 1.9 μm) at a flow rate of 300 nL min-1. Data were acquired in DDA-PASEF mode over an m/z range of 100–1700, with ion mobility separation (1/K0 0.75–1.50 V·s·cm-2). MS/MS spectra were acquired with collision energies of 20–59 eV, aPeptide digestion was performed using the FASP protocol with Microcon PL-10 filters. After three rounds of buffer exchange with 8 M urea in 100 mM Tris-HCl (pH 8.0), proteins were reduced with 10 mM DTT at 37 °C for 30 min and alkylated with 30 mM iodoacetamide at 25 °C for 45 min in the dark. Samples were then exchanged into digestion buffer (30 mM Tris-HCl, pH 8.0) and digested with trypsin (enzyme-to-protein ratio 1:50) at 37 °C for 12 h. The filtrates were collected, combined after washing the filter twice with 15% ACN, and vacuum-dried. Phosphopeptides were enriched using home-made TiO2 microcolumns (peptide: TiO2 = 1:10). Peptides were dissolved in loading buffer (1 M glycolic acid, 80% ACN, 5% TFA), loaded twice, washed with loading buffer followed by washing buffer (80% ACN, 1% TFA), and sequentially eluted with 2 M ammonium hydroxide and 1 M ammonium hydroxide in 30% ACN. Eluates were vacuum-dried. nd dynamic exclusion was enabled.", "projectTags" : [ ], "keywords" : [ "Shade avoidance; sucrose; phya; tor; leaf development" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-21", "updatedDate" : "2026-03-20", "submissionDate" : "2026-03-20", "downloadCount" : 8, "avgDownloadsPerFile" : 1.1428572, "botCount" : 4, "hubCount" : 4, "organicCount" : 0, "percentile" : 1, "yearlyDownloads" : [ { "year" : "2026", "count" : 8 } ], "submitters" : [ "Jiayu Wang" ], "labPIs" : [ "Lin Li" ], "affiliations" : [ "University of Fudan" ], "instruments" : [ "ultraflex", "Q Exactive" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Arabidopsis thaliana (Mouse-ear cress)", "shoot", "leaf" ], "organisms" : [ "Arabidopsis thaliana (mouse-ear cress)" ], "organismsPart" : [ "Leaf", "Shoot" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Proteogenomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "HT-SKY-LL-WJY-20250106-phyA-vitro-pho_Slot1-68_1_5122_6.1.213.pdResult", "HT-SKY-LL-WJY-20250106-phyA-vitro_Slot1-69_1_5123_6.1.213.pdResult", "Exploris05530-SKY-LL-WJY-20250317-phyA-pho.pdResult", "Exploris05530-SKY-LL-WJY-20250317-phyA-pho.raw", "HT-SKY-LL-WJY-20250106-phyA-vitro-pho_Slot1-68_1_5122.d.zip", "HT-SKY-LL-WJY-20250106-phyA-vitro_Slot1-69_1_5123.d.zip" ], "highlights" : { } }, { "accession" : "PXD075878", "title" : "The translatome of quiescent Plasmodium falciparum gametocytes reveals parasite pyridoxal 5’-phosphate (PLP) biosynthesis is essential for efficient mosquito stage development", "projectDescription" : "The ability of Plasmodium falciparum gametocytes to remain quiescent within the vertebrate host but poised for rapid onward development in the mosquito is an adaptation essential to maximise the onward spread of malaria. In this dormant state, mature infectious stage V gametocytes are largely unaffected by most antimalarial drugs and our limited understanding of how gametocytes prepare for mosquito transmission has hindered the identification of new molecular targets for transmission-blocking therapeutics. In this study, we move beyond the total proteome of gametocytes and define the translatome of mature stage V gametocytes using L-azidohomoalanine incorporation into nascent proteins, click chemistry purification and proteomic analysis. We identify the proteins and pathways that gametocytes sustain in preparation for transmission during this dormant period and through genetic disruption, we validate this approach by demonstrating the importance of parasite pyridoxal 5’-phosphate biosynthesis for mosquito transmission.", "dataProcessingProtocol" : "DIA data from whole proteome samples were processed using DIA-NN (version 1.8.1)14 in library-free mode. The analysis used the Plasmodium falciparum 3D7 protein database downloaded from UniProt on 15 December 2023, which included 5374 proteins and 246 common contaminants. Deep learning-based prediction of spectra and retention times was enabled. Trypsin specificity was applied, allowing one missed cleavage, with carbamidomethylation of cysteine as a fixed modification and oxidation (M), N-terminal acetylation, and N-terminal excision set as variable modifications, with up to two variable modifications per peptide. Match-between-runs (MBR) was enabled, and quantification was performed using the \"Robust LC (high precision)\" strategy. Heuristic protein inference was disabled, and both mass and MS1 accuracy were set to 0. DDA data from the click pulldown samples were analysed using the FragPipe suite (version 5.0.0) with the built-in “LFQ MBR” (Label-Free Quantification with Match-Between-Runs) workflow. Raw .d files were processed with MSFragger (version 3.8)15, using strict trypsin digestion with up to two missed cleavages allowed, and a precursor ion tolerance of 20 ppm. Carbamidomethylation of cysteine was set as a fixed modification, while oxidation (M) and N-terminal acetylation were defined as variable modifications. Trimming of N-terminal methionine residues was enabled. The same P. falciparum 3D7 database used for DIA-NN was also used here, supplemented with 50% decoy sequences and common contaminants via the inbuilt FragPipe utility. Peptide-spectrum matches were validated using Percolator (version 3.5)16, and protein inference and FDR filtering were performed with Philosopher (version 5.0.0)17. Label-free quantification was conducted using IonQuant (version 5.0.0)18, with match-between-runs enabled and the minimum number of ions for quantification set as 1.", "sampleProcessingProtocol" : "The frozen gametocyte pellets were lysed in cold lysis buffer consisting of PBS supplemented with 1% Triton X-100, 0.01% SDS, and EDTA-free HaltTM protease inhibitor cocktail. The lysates were incubated on ice for one hour, then sonicated using an ultrasonic bath (Grant Instruments Cambridge Ltd) at full power for 30 seconds, repeated three times with 30-second intervals. Following sonication, samples were centrifuged at 16,000 × g for 30 minutes at 4°C, and the soluble fraction was collected. Protein concentration was determined using the Pierce BCA Protein Assay Kit. The soluble fractions were either directly processed for whole proteome analysis or used for click chemistry pulldown enrichment. The soluble fraction was incubated at room temperature for one hour on a shaker with a click reaction mix composed of 100 µM PEG4 carboxamide-propargyl biotin (Thermo Fisher Scientific), 1 mM copper sulfate (Scientific Laboratory Supplies), 1 mM of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Promega UK Ltd), and 100 µM tris((1-benzyl-4-triazolyl)methyl)amine (TBTA, Cambridge Bioscience). The reaction was quenched with 5 mM EDTA. Proteins were precipitated with acetonitrile and resuspended in 50 mM HEPES buffer (pH 8.0) containing 0.2% SDS, followed by incubation with NeutrAvidin agarose beads (ThermoFisher Scientific) for one hour at room temperature on a shaker. The beads were washed four times with the same buffer before proceeding to on-bead digestion. All protein samples, including whole proteome and enriched fractions, were reduced with 10 mM TCEP and alkylated with 40 mM 2-chloroacetamide (CAA), followed by overnight digestion at 37°C using sequencing-grade modified trypsin (Promega UK Ltd).", "projectTags" : [ ], "keywords" : [ "Plasmodium falciparum", "Translatome", "Malaria" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-25", "updatedDate" : "2026-03-19", "submissionDate" : "2026-03-19", "downloadCount" : 5, "avgDownloadsPerFile" : 0.45454547, "botCount" : 1, "hubCount" : 4, "organicCount" : 0, "percentile" : 0, "yearlyDownloads" : [ { "year" : "2026", "count" : 5 } ], "submitters" : [ "Jack Houghton" ], "labPIs" : [ "Michael Delves" ], "affiliations" : [ "London School of Hygine and Tropical Medicine" ], "instruments" : [ "timsTOF HT" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Plasmodium falciparum", "malaria" ], "organisms" : [ "Plasmodium falciparum" ], "organismsPart" : [ ], "diseases" : [ "Malaria" ], "references" : [ "null--pubMed:0--doi: 10.64898/2026.03.24.713170" ], "experimentTypes" : [ "Data-dependent acquisition", "Data-independent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "translatome-combined_protein.tsv", "JH-EAdS-WT-3_S3-B11_1_2994.d.zip", "translatome-combined_peptide.tsv", "JH-ES-C-2_S4-F2_1_1454.d.zip", "JH-EAdS-WT-1_S3-B9_1_2992.d.zip", "total-gametocyte-proteome-report.pr_matrix.tsv", "JH-ES-B_S4-F1_1_1451.d.zip", "total-gametocyte-proteome-report.pg_matrix.tsv", "JH-EAdS-WT-2_S3-B10_1_2993.d.zip", "JH-ES-A_S4-E12_1_1450.d.zip" ], "highlights" : { } }, { "accession" : "PXD075871", "title" : "IP-MS analysis of RLF/ZFP292/ZFP654-FLAG", "projectDescription" : "We performed immunoprecipitation followed by LC-MS/MS (IP-MS) in mouse embryonic stem cells in which endogenous RLF, ZFP292 or ZFP654 was tagged with FLAG. This approach enabled the identification of proteins associated with RLF/ZFP292/ZFP654. The dataset provides a resource to investigate the composition of RLF/ZFP292.ZFP654-associated chromatin regulatory complexes.", "dataProcessingProtocol" : "Raw MS data were processed and searched against the mouse proteome database (UniProtKB, released January 2025) using MaxQuant software (v2.6.7.0) for Q Exactive Plus data. Significant interacting proteins were calculated and defined by p-value < 0.05 and |Log2(fold change)| > 1 using Razor + unique peptide counts.", "sampleProcessingProtocol" : "Approximately 3 × 10^7 cells were cultured in one 15-cm dish for DTSSP-crosslinked samples. Cells were crosslinked with 0.5 mM DTSSP (Wako, 342-09131) diluted in 0.1 M NaCl CSK buffer (10 mM PIPES, pH 7.0; 100 mM NaCl; 300 mM sucrose; 1 mM MgCl2; 1 mM EGTA; 0.1% Triton X-100) for 35 min. Crosslinking was quenched with 15 mM Tris-HCl (pH 7.5), concurrently with permeabilization. Permeabilized cells were treated with 25 U/ml KANEKA endonuclease (KANEKA, KEN02100) at 4°C for 30 min to digest all nucleic acids. The NaCl concentration was then increased to 0.5 M to stop the digestion and solubilize proteins. The lysate was centrifuged at 16,000 × g for 20 min, and the supernatant was used as input for immunoprecipitation. The input was incubated with anti-FLAG M2 affinity gel beads (Millipore, A2220) at 4°C for 2 h. Beads were washed with 0.5 M NaCl CSK buffer, and bound proteins were eluted using 3× FLAG peptide (F4799, Millipore). Eluted samples were separated by SDS-PAGE and cut into five gel slices, followed by in-gel trypsin digestion. LC-MS/MS was performed using reverse-phase chromatography coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific).", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-21", "updatedDate" : "2026-03-19", "submissionDate" : "2026-03-19", "downloadCount" : 3, "avgDownloadsPerFile" : 0.024590164, "botCount" : 1, "hubCount" : 2, "organicCount" : 0, "percentile" : 0, "yearlyDownloads" : [ { "year" : "2026", "count" : 3 } ], "submitters" : [ "Takamasa Ito" ], "labPIs" : [ "Chikashi Obuse" ], "affiliations" : [ "Laboratory of Genome Structure and Function Department of Biological Sciences Graduate School of Science The University of Osaka" ], "instruments" : [ "Q Exactive Plus" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "embryonic stem cell", "cell culture", "Mus musculus (Mouse)" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Cell culture", "Embryonic stem cell" 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Many clinically important drug targets require expression in eukaryotic systems-such as mammalian, yeast, or insect cells-rather than prokaryotic hosts. This requirement limits the feasibility of high-throughput isotopic labeling and poses challenges for obtaining uniformly isotope-labeled proteins suitable for NMR analysis. While several enrichment strategies have been developed, no broadly applicable enrichment platform has emerged for eukaryotic expression systems. In this study, we introduce Cupriavidus necator as an alternative biological source for 15N and 13C isotopic enrichment to support protein production in eukaryotic systems. To evaluate this approach, we selected the kinase domain of EPHA2, a receptor tyrosine kinase implicated in colorectal cancer progression and an important target for therapeutic inhibitor development. Isotopic incorporation was quantified using liquid chromatography-mass spectrometry (LC-MS), revealing enrichment levels of 79% for 15N and 69% for 13C. These results demonstrate that Cupriavidus necator can serve as a robust and flexible platform for generating isotopically enriched biomolecules compatible with eukaryotic protein expression, thereby enabling NMR investigations of disease-relevant protein targets.", "dataProcessingProtocol" : "Database searches The resulting files were recalibrated, and database searches were performed using the MaxQuant search engine (1.5.3.8) against a combined database containing the host cell proteome (Spodoptera, tax_id:7106, downloaded 17-08-2017), the construct sequence and common contaminants. For database searches, precursor mass tolerance was set to 20 ppm in the primary analysis and 4.5 ppm in the full search, with fully tryptic cleavages with up to two missed cleavages. Oxidation of methionines residues (+15.995) and acetylation of protein N-terminal (+42.011) were set as variable modifications and carboxyamidomethylation (+57.021) as a fixed modification. Peptide and protein FDR was set to 0.01. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Perez-Riverol et al. 2025). Anonymous reviewer access is available upon request. Calculation of isotopic labeling efficiency (15N, 13C and 15N+13C labeling) First, target-protein derived peptides were selected in unlabeled samples based on high-quality, unambiguous fragment spectra for identification, peptide intensity and chromatographic elution. In addition, all peptides were manually curated to ensure no interfering other peaks were present within the expected isotopic envelope. A list of 20 high-quality candidate peptides were manually selected for further processing. The incorporation of 15N and 13C was determined by comparing the measured isotopic envelope distributions to the respective theoretical mass of the unlabeled and heavy isotope-labeled peptide, which is a common strategy in HDX-MS (Masson et al. 2019). In brief, extracted ion chromatograms were created for high quality peptide candidates (peptide identification and protein FDR 0.01, intensity >10e4, no interfering isotopic envelopes by other compounds in control samples). Then, peak areas were integrated for all assigned isotopic peaks for each peptide based on the maximum possible incorporation for 15N and 13C, and intensity-weighted centroids were calculated. All assigned ion spectra were manually inspected and revised, and candidates with interfering isotopic envelopes from other compounds in the labelled samples were discarded. 15N/13C incorporation was then calculated by comparing the difference in intensity-weighted centroids compared to the unlabeled control and the fully labeled theoretical mass. At least 5 unique peptides were used for calculation of the 15N/13C uptake. The full code is available on PRIDE together with the raw data.", "sampleProcessingProtocol" : "Digestion In-gel Gel bands were digested with the In-Gel Tryptic Digestion kit (Thermo Fisher Scientific) with minor adaptations. The excisions were destained, reduced and alkylated according to the manufacturer’s protocol and digested overnight with trypsin (SERVA) at room temperature. The digested peptides were transferred to a clean tube and the solvent was evaporated in a speed vac (Eppendorf). The dried peptides were stored at -20°C until LC-MS analysis. In-solution About 50 µg protein were loaded per filter unit (Microcon-10, Merck Millipore). After addition of 200 µL UA solution (6.5 m Urea in 0.1 m Tris/HCl pH 8.5) to the filter the sample was centrifuged at 14 000 × g for 20 min. This step was repeated twice and the flow through was discarded carefully. The sample was incubated at room temperature with 100 µL UA solution containing 10 mm dithiothreitol (DTT) using a thermomixer for 30 min in the dark. The filter unit was centrifuged at 14 000 × g for 20 min and the flow through was discarded carefully. Subsequently 100 µL of UA solution containing 95 mm Iodoacetamide (IAA) was added and incubated at room temperature using a thermomixer for 30 min in the dark. The filter unit was then centrifuged at 14 000 × g for 20 min and the flow through was discarded. The filter was washed three times with 100 µL of UB solution (6.5 m Urea in 0.1 m Tris/HCl, pH 8) and centrifuged at 14 000 × g for 20 min. The filter unit was incubated with 40 µL of UB containing Lys-C in a ratio of 1:50 in the dark at room temperature overnight. The unit was transferred to a new collection tube and 100 µL of ABS solution containing trypsin in a ratio 1:100 were added and the unit was incubated in the dark at room temperature overnight. The unit was centrifuged at 14 000 × g for 15 min. Then 50 µL of 0.5 m NaCl was added and the filter unit was centrifuged at 14 000 × g for 15 min. The filtrate was acidified with 0.1% TFA and was desalted by C-18 ZipTips (Merck Millipore) according to the manufacturers protocols and dried in a SpeedVac. The dried peptides were stored at -20°C until LC-MS analysis. LC-MS data acquisition The dried peptide fractions were dissolved in 5% acetonitrile with 0.1% formic acid, and subsequently loaded on reverse phase columns (trapping cartridge: 5 µm C18-beads, L = 5 mm, inner diameter = 100 µm; Thermo Fisher Scientific, Bremen); analytical column: ReproSil-Pur C18-AQ 1.8 µm beads, 75 µm inner diameter, L = 50 cm (PepSep, Marslev, Denmark) using a nano-HPLC (Bruker nanoElute). Eluted peptides were separated over a 90 min gradient of water (buffer A: water with 0.1% formic acid) and acetonitrile (buffer B: acetonitrile with 0.1% formic acid). All LC-MS-grade solvents were purchased from Fluka. Gradients were ramped from 2% to 35% B in 90 min at flowrates of 300 nL/min. Peptides eluting from the analytical column were ionized online using a Bruker CaptiveSpray ESI-source and analyzed in a Bruker Impact-II mass spectrometer. Mass spectra were acquired over the mass range 150–2200 m/z, and sequence information was acquired by a computer-controlled, dynamic method with a fixed cycle time of 3 s and an intensity-dependent acquisition speed for MS/MS-spectra between 8 and 20 Hz (instant expertise-mode) of the candidate ions.", "projectTags" : [ ], "keywords" : [ "Cupriavidus necator", "Nmr", "Isotope labeling" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-26", "updatedDate" : "2026-03-18", "submissionDate" : "2026-03-18", "downloadCount" : 5, "avgDownloadsPerFile" : 0.5, "botCount" : 0, "hubCount" : 5, "organicCount" : 0, "percentile" : 0, "yearlyDownloads" : [ { "year" : "2026", "count" : 5 } ], "submitters" : [ "Julian Langer" ], "labPIs" : [ "Julian Langer" ], "affiliations" : [ "MPI of Biophysics and MPI of Brain Research" ], "instruments" : [ "impact II" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Nosema spodopterae" ], "organisms" : [ "Nosema spodopterae" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "1554_rawdata.zip", "SaGande_labelPeptides.R", "SDRF_Gande_et_al.xlsx", "checksum.txt", "1554_results_txt.zip", "SaGande_MQ.R", "1734_results_mgf.zip", "1687_results_txt.zip", "1734_rawdata.zip", "1687_rawdata.zip" ], "highlights" : { } }, { "accession" : "PXD075811", "title" : "Evaluation of protein artifacts in tissue cryopreservation", "projectDescription" : "This dataset presents proteomic analyses validating the spatial proteomics technology, NicheProt. NicheProt is a 3D optical microscopy–guided, photobleaching-mediated cell barcoding approach for isolating intact cell types from defined microanatomical niches. Here, we evaluate whether tissue cryopreservation introduces protein artifacts by comparing proteomic profiles of mouse spleens processed immediately after fixation with those cryopreserved in 100% D-fructose at −80 °C prior to extraction. Our results demonstrate that D-fructose–based cryopreservation preserves protein integrity without introducing significant artifacts.", "dataProcessingProtocol" : "Raw MS files were processed using label-free quantification (LFQ) and searched against a Swissprot mouse database (SP_mouse_2024_06; 17208 entries) using the Sequest HT node in Proteome Discoverer Software (version 3.0.0.757). Peptide and protein identifications are filtered at a 1% false discovery rate (FDR). Proteomics downstream data analysis and visualization were performed using R (version 2024.09.1+394).", "sampleProcessingProtocol" : "Fixed mouse spleens were bisected and assigned to either cryopreservation or control groups. Cryopreserved samples were incubated in 100% (w/v) D-fructose and subsequently thawed prior to tissue dissociation, whereas control samples were dissociated into single-cell suspensions on the day of harvest. Cells were lysed at 95 °C for 30 minutes followed by 65 °C for 1 hour in lysis buffer (1× RIPA buffer, 1× complete protease inhibitor, 1× PhosSTOP phosphatase inhibitor cocktail, and 3% [w/v] SDS). Extracted proteins were digested overnight on S-Trap columns with trypsin. LC–MS/MS analysis was performed using a Vanquish Neo UHPLC system equipped with a trap column (PepMap C18, 300 µm × 5 mm) and an analytical column (IonOpticks Ultimate C18, 1.7 µm, 75 µm × 25 cm), coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "10.6019/PXD075811", "submissionType" : "COMPLETE", "publicationDate" : "2026-03-19", "updatedDate" : "2026-03-18", "submissionDate" : "2026-03-18", "downloadCount" : 343, "avgDownloadsPerFile" : 24.5, "botCount" : 317, "hubCount" : 19, "organicCount" : 7, "percentile" : 12, "yearlyDownloads" : [ { "year" : "2026", "count" : 343 } ], "submitters" : [ "Steve Seung-Young Lee" ], "labPIs" : [ "Steve Seung-Young" ], "affiliations" : [ "Department of Pharmaceutical Sciences, University of Illinois College of Pharmacy,Chicago,IL,USA Richard and Loan Hill Department of Biomedical Engineering, University of Illinois College of Engineering,Chicago, IL,USA University of Illinois Cancer Center,Chicago, IL,USA" ], "instruments" : [ "Orbitrap Exploris 480" ], "softwares" : [ ], "quantificationMethods" : [ "Absolute quantification" ], "sampleAttributes" : [ "spleen", "Mus musculus (Mouse)", "lymphocyte" ], "organisms" : [ "Mus musculus", "Mus musculus (mouse)" ], "organismsPart" : [ "Spleen", "Lymphocyte" ], "diseases" : [ "Control", "Cryopreservation" ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "Spleen N/a Mus musculus Ac=ms:1001251;nt=trypsin Male Nt=carbamidomethyl;ac=unimod:4;ta=c;mt=fixed 1 Data-dependent acquisition Control 2 3 Nt=label free sample Nt=oxidation;mt=variable;ta=m;ac=unimod:35; nt=acetylation;mt=variable;ac=unimod:1 4 Cryopreservation Proteomic profiling by mass spectrometry 5 6-8 weeks Ac=ms:1003028; nt= orbitrap exploris 480 0.02 da Lymphocyte 10 ppm", "projectFileNames" : [ "CE3_2025Apr04_P25-047_UIC_Lee_CS10_2ul.raw", "CE3_2025Apr04_P25-047_UIC_Lee_CS11_2ul.raw", "CE3_2024Jun03_UIC_P24035_CS2.mzML", "CE3_2025Apr04_P25-047_UIC_Lee_CS13_2ul.raw", "CE3_2025Apr04_P25-047.sdrf.tsv", "CE3_2024Jul31_11_P24-095_CS7.raw", "CE3_2025Apr04_P25-047_UIC_Lee_CS12_2ul.raw", "checksum.txt", "CE3_2024Jul31_09_P24-095_CS6.raw", "CE3_2024Jul31_05_P24-095_CS4.raw", "CE3_2025Apr04_P25-047_UIC_Lee_CS9_2ul.raw", "CE3_2024Jun03_UIC_P24035_CS2.mzid", "CE3_2025Apr04_P25-047_UIC_Lee_CS8_2ul.raw", "CE3_2024Jul31_07_P24-095_CS5.raw" ], "highlights" : { } }, { "accession" : "PXD075769", "title" : "Evaluation of protein artifacts in the NicheProt workflow", "projectDescription" : "This dataset contains proteomic analyses that validate the spatial proteomics technology, NicheProt. NicheProt is a 3D optical microscopy–guided, photobleaching-mediated cell barcoding approach designed to isolate intact cell types from specific microanatomical niches. We evaluate the proteomic profiles of samples processed using the multi-step NicheProt workflow and demonstrate that it does not introduce detectable protein artifacts.", "dataProcessingProtocol" : "Raw MS files were processed using label-free quantification (LFQ) and searched against a Swissprot mouse database (SP_mouse_2024_06; 17208 entries) using the Sequest HT node in Proteome Discoverer Software (version 3.0.0.757). Peptide and protein identifications are filtered at a 1% false discovery rate (FDR). Proteomics downstream data analysis and visualization were performed using R (version 2024.09.1+394).", "sampleProcessingProtocol" : "Fixed mouse spleens were bisected and designated for control or NicheProt processing. Control samples consisted of splenocytes/lymphocytes dissociated from cryopreserved spleen macrosections (400 μm thick), whereas NicheProt samples were dissociated from macrosections processed through the multi-step NicheProt workflow, including immunofluorescent staining, tissue clearing, photobleaching-based barcoding, and reverse tissue clearing. Cells were subsequently isolated and lysed at 95 °C for 30 minutes followed by 65 °C for 1 hour in lysis buffer (1× RIPA buffer, 1× complete protease inhibitor, 1× PhosSTOP phosphatase inhibitor cocktail, and 3% [w/v] SDS). Extracted proteins were digested overnight on S-Trap columns with trypsin. LC–MS/MS analysis was performed using a Vanquish Neo UHPLC system equipped with a trap column (PepMap C18, 300 µm × 5 mm) and an analytical column (IonOpticks Ultimate C18, 1.7 µm, 75 µm × 25 cm), coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "10.6019/PXD075769", "submissionType" : "COMPLETE", "publicationDate" : "2026-03-19", "updatedDate" : "2026-03-18", "submissionDate" : "2026-03-18", "downloadCount" : 36, "avgDownloadsPerFile" : 3.6, "botCount" : 24, "hubCount" : 10, "organicCount" : 2, "percentile" : 2, "yearlyDownloads" : [ { "year" : "2026", "count" : 36 } ], "submitters" : [ "Steve Seung-Young Lee" ], "labPIs" : [ "Steve Seung-Young" ], "affiliations" : [ "Department of Pharmaceutical Sciences, University of Illinois College of Pharmacy,Chicago,IL,USA Richard and Loan Hill Department of Biomedical Engineering, University of Illinois College of Engineering,Chicago, IL,USA University of Illinois Cancer Center,Chicago, IL,USA" ], "instruments" : [ "Orbitrap Exploris 480" ], "softwares" : [ ], "quantificationMethods" : [ "Absolute quantification" ], "sampleAttributes" : [ "spleen", "Mus musculus (Mouse)", "lymphocyte" ], "organisms" : [ "Mus musculus", "Mus musculus (mouse)" ], "organismsPart" : [ "Spleen", "Lymphocyte" ], "diseases" : [ "Control", "Nicheprot" ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "Spleen N/a Mus musculus Ac=ms:1001251;nt=trypsin Data dependent acquisition Male Nt=carbamidomethyl;ac=unimod:4;ta=c;mt=fixed 1 Control 2 3 Nt=label free sample Nt=oxidation;mt=variable;ta=m;ac=unimod:35; nt=acetylation;mt=variable;ac=unimod:1 Lymphocytes Proteomic profiling by mass spectrometry Nicheprot 6-8 weeks Ac=ms:1003028; nt= orbitrap exploris 480 0.02 da 10 ppm Nt=oxidation;mt=variable;ta=m;ac=unimod:35 ; nt=acetylation;mt=variable;ac=unimod:1", "projectFileNames" : [ "checksum.txt", "CE3_2024Oct08_13_P24-162_ES3.raw", "CE3_2024Oct08_05_P24-162_ES1.raw", "CE3_2024Oct08_09_P24-162_ES2.raw", "CE3_2024Oct08_15_P24-162_ES6.raw", "CE3_2024Oct08_05_P24-162_ES1-ES6.mzML", "CE3_2024Oct08_11_P24-162_ES5.raw", "CE3_2024Oct08_P24-162.sdrf.tsv", "CE3_2024Oct08_05_P24-162_ES1-ES6.mzid", "CE3_2024Oct08_07_P24-162_ES4.raw" ], "highlights" : { } }, { "accession" : "PXD075759", "title" : "Host-adapted enzymatic deconstruction of acetylated xylan enables mutualistic colonization of monocot roots", "projectDescription" : "Intracellular accommodation of beneficial fungi requires host cell wall remodeling that avoids excessive immune activation. The root endophyte Serendipita indica, a generalist mutualist capable of colonizing both monocot and dicot plants, employs a monocot-specific enzymatic module to deconstruct acetyl-xylan, the dominant hemicellulose of grasses. Central to this module are the glycoside hydrolase SiGH11, which releases acetylated xylooligosaccharides, and SiAXE, a previously uncharacterized SGNH-like acetyl-xylan esterase that sequentially removes acetyl groups from soluble XOS. Both enzymes are co-expressed within a monocot-enriched transcriptional program that also includes sugar transporters and metabolic regulators. Their coordinated activity, together with co-expressed exo-enzymes, promotes efficient xylan hydrolysis while limiting the prolonged accumulation of immunogenic damage-associated molecular patterns (DAMPs). Functional genetics demonstrated that SiAXE is required for sustained intracellular growth in monocot roots: its deletion impaired colonization, whereas overexpression transiently accelerated entry but provoked immune responses, underscoring the importance of temporal regulation and enzyme coordination for immune-compatible colonization. These findings provide mechanistic insight into an immune-compatible fungal strategy for host cell wall remodeling and reveal how a broadly colonizing mutualist has repurposed ancestral saprotrophic enzymes into specialized host-adapted modules that balance nutrient acquisition with immune modulation.", "dataProcessingProtocol" : "Samples were analyzed in Spectronaut 19 (Biognosys) in directDIA mode using the Serendipita indica DSM 11827 protein database (downloaded 12.09.25 at fungi.ensembl.org). Seach parameters were following recommended standard settings with minimum and maximum fragement filters set to six and 12, respectively. Afterwards, analysis of results was performed in Perseus 1.6.15. Low-intensity quantifications were converted to NA, followed by log2 transformation of remaining LFQ values, filtering on data completeness in at least one replicate group and FDR-controlled t-tests between the two groups of interest. Samples were filtered for NA in mycelium protein samples and differential protein abundance visualized in a volcano plot of log2FC against -log10-transformed p-values.", "sampleProcessingProtocol" : "Apoplastic fluid (AF) was isolated from barley seedlings as described previously (Saake et al, 2025 https://doi.org/10.1111/nph.70022). Germinated barley seedlings were inoculated with S. indica on 1/10 PNM medium and per replicate, 40 barley seedlings were harvested at 14 dpi, which yielded approximately ~0.5 ml AF per replicate. Harvested roots were surface washed thoroughly in ice-cold water to remove external fungal material, cut into pieces of 2 cm, transferred to ice cold water and vacuum infiltrated (five cycles of15 min 250 mbar, 1.5 min ATM). Then, the roots were gently dried on tissue paper and transferred into a 20 ml syringe in a 50 ml falcon tube, centrifuged for 15 min at 4 °C, 2000 rpm (lowest de- and acceleration) to collect AF in the falcon tube. The isolated AF was stored at -20°C. Proteome analysis was done for the following samples: AF from barley colonized by Serendipita indica expressing GFP (GFP-2), SiAXE (AXE-3). As control, protein was extracted from mycelium of GFP-2 grown in axenic culture by the following protocol: a 100 mL starter culture of CM medium was inoculated with chlamydospores and incubated for one week at 28°C while shaking at 120 rpm. The mycelium was harvested using Miracloth (22-25 µm pore size) and washed with sterile MQ-H2O containing 0.9 % NaCl. The washed mycelium was resuspended in 100 mL fresh CM medium and homogenized with a blender. The resulting mycelial fragments were incubated at 28 °C while shaking at 130 rpm to allow regeneration. After three days, the mycelium was harvested using Miracloth (22-25 µm pore size). The mycelium was washed with 0.9 % NaCl, blotted dry on tissue paper and frozen in liquid nitrogen for protein extraction with the following method: S. indica mycelium was ground with mortar and pestle under liquid N2 to a fine powder and 200 mg FW transferred per sample to a 2 ml tube. Then, 500 µl of pre-cooled TKMES extraction buffer (100 mM tricine; pH=7.5, 10 mM KCL, 10 mM MgCl2, 1 mM EDTA, 10% w/v sucrose, freshly added 0.2 % v/v Triton X-100, 1 mM DTT, 100 µg ml-1 PMSF, 1x protease inhibitor cocktail (Sigma Aldrich; #P9599) was added and vortexed vigorously for 20 seconds. The homogenate was centrifuged at 10000 g for 10 minutes at 4°C to pellet cell debris and the supernatant harvested. For AF and mycelium samples, protein amount was determined with Bradford reagent (Bio-Rad) and 5 µg total protein precipitated with acetone, prior to reduction with DTT and alkylation of cysteine residues with chloroacetamide. Then, protein were subjected to on-bead digestion on Sera-Mag beads with trypsin and endoproteinase Lys-C and desalted using SDB-RPS StageTips and prepared for label-free quantitative proteomics. Protein mass spectrometry of digested peptides was done on an Orbitrap Exploris 480 mass spectrometer coupled to a Vanquish neo in trap-and-elute setup (Thermo Scientific). Samples were loaded onto a 2 cm precolumn (Acclaim Pepmap 100, Thermo Scientific) with a flow of 10 µl min-1 of eluent A (0.1% formic acid) before in-line flushed onto an Auroa Ultimate column (25 cm length, 75 µm inner diameter, Ionopticks). Peptides were chromatographically separated with an initial flow rate of 400 nL min-1 and the following gradient: initial 4% B (0.1% formic acid in 80 % acetonitrile), up to 8.5 % in 4 min. Then, flow was reduced to 300 nl/min and B increased to 35% B in 75 min, and up to 98% solvent B within 1.0 min while again increasing the flow to 400 nl/min, followed by column wash and reequilibration to initial condition.MS1 scans were acquired from 399 m/z to 1001 m/z at 30k resolution. Maximum injection time was set to 54 ms and the AGC target to 100%. MS2 scans ranged from 400 m/z to 1000 m/z and were acquired at 30 k resolution with a maximum injection time of 54 ms and an AGC target of 1000%. DIA scans covering the precursor range from 400-1000 m/z and were acquired in 30 x 20 m/z windows with an overlap of 1 m/z. All scans were stored as centroid", "projectTags" : [ ], "keywords" : [ "Symbiosis", "Hemicellulose remodeling", "Host immune modulation", "Carbohydrate-active enzymes (cazymes)", "Acetyl-xylan esterase", "Plant-microbe interactions" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-20", "updatedDate" : "2026-03-17", "submissionDate" : "2026-03-17", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Proteomics Facility" ], "labPIs" : [ "Prof. Dr. Alga Zuccaro" ], "affiliations" : [ "Institute for Plant Sciences, University of Cologne, 50674 Cologne, Germany" ], "instruments" : [ "Orbitrap Exploris 480" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Serendipita indica DSM 11827", "protein secretion" ], "organisms" : [ "Serendipita indica dsm 11827" ], "organismsPart" : [ "Protein secretion" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-independent acquisition" ], "sdrf" : "", "projectFileNames" : [ "E2_4597_16.raw", "E2_4597_08.raw", "4597_AXE_GFP_FASTA.fasta", "4597_PG_Report.setup.txt", "E2_4597_07.raw", "4597_PG_Report.tsv", "E2_4597_13.raw", "E2_4597_15.raw", "4597_Name.tsv", "E2_4597_11.raw", "checksum.txt", "4597.sne", "E2_4597_06.raw", "E2_4597_14.raw", "E2_4597_12.raw", "E2_4597_10.raw" ], "highlights" : { } }, { "accession" : "PXD075747", "title" : "Small Molecule Guided Small Molecule Guided Photocatalytic Proteomics Profiling of Amyloid Deposits in Hippocampal and Cortical Alzheimer’s Disease TissuesPhotocatalytic Proteomics Profiling of Amyloid Deposits in Hippocampal and Cortical Alzheimer’s Disease Tissues", "projectDescription" : "he spatiotemporal progression of amyloid pathology in Alzheimer's disease (AD) follows a characteristic pattern, spreading from the cortex to the hippocampus brain regions. However, analytical methods for comparative profiling of amyloid plaque composition between these two regions are limited. Herein, we developed a small molecule guided method to selectively label, enrich, profile and compare amyloid interactome in cortical and hippocampal regions of AD brain tissue. We embarked on rational design of probes to transform Congo Red derivatives from amyloid chromophore to singlet fluorescent sensor, and finally to triplet photocatalytic labeling probe. While retaining the amyloid binding selectivity, P5 outperformed other probes in photocatalytic labeling of recombinant amyloid proteins and amyloid deposits from AD mouse brain tissues. We applied P5 to selectively labeling and enrichment of amyloid plaques in hippocampus and cortex, respectively. The robustness of our methodology was confirmed by the consistent identification of established AD biomarkers (e.g. APP, ApoE) in both regions. Subsequent comparative proteomics not only demonstrated the critical involvement of the mitophagy-lysosome axis in AD pathogenesis, but also uncovered a previously unrecognized region-specific functional divergence. Proteomic profiles distinguished that AD’s cortex primarily involves upstream mitophagy, whereas AD’s hippocampus actively triggers downstream lysosomal degradation. Overall, we report a small-molecule-based photocatalytic proteomic profiling method to resolve amyloid deposits and elucidate their region-specific interactome heterogeneity.", "dataProcessingProtocol" : "The tryptic peptides were dissolved in solvent A, directly loaded onto a home-made reversed-phase analytical column (25-cm length, 100 μm i.d.). The mobile phase consisted of solvent A (0.1% formic acid, 2% acetonitrile/in water) and solvent B (0.1% formic acid in acetonitrile). Peptides were separated with following gradient: 0-14 min，6%-24% B；14-16 min，24%-35% B；16-18 min，35%-80% B；18-20 min，80% B, and all at a constant flow rate of 500 nl/minon a NanoElute UHPLC system (Bruker Daltonics). The peptides were subjected to capillary source followed by the timsTOF Pro mass spectrometry. The electrospray voltage applied was 1.75 kV. Precursors and fragments were analyzed at the TOF detector. The timsTOF Pro was operated in data independent parallel accumulation serial fragmentation (dia-PASEF) mode. The full MS scan was set as 300-1500 (MS/MS scan range) and 20PASEF (MS/MS mode) - MS/MS scans were acquired per cycle. The MS/MS scan range was set as 400-850 and isolation window was set as 7 m/z.", "sampleProcessingProtocol" : "Brain sections from APP/PS1 mice (experimental group) and age-matched wild-type C57BL/6 mice (negative control group) were subjected to antigen retrieval in sodium citrate buffer (10.0 mM, pH = 6.0) at 95 °C for 20 minutes, followed by sequential procedures: blocking with endogenous biotin blocking solution at room temperature for 30 minutes, staining with P5 probe (150.0 μM) in the dark for 30 minutes. After three PBS washes, sections were incubated with amino-PEG2-biotin (10.0 mM) in darkness for 10 minutes, then transferred to white light illumination (25 mW·cm-2) for 20 minutes. Immediately after illumination, sections were rinsed with PBS and lysed with 6.0 M guanidine hydrochloride containing 1% protease inhibitors and 1% protein phosphatase inhibitors. The lysate was diluted to a final concentration of 1.0 M guanidine hydrochloride using 50.0 mM NH4HCO3, then incubated with streptavidin-agarose resin (Catalog No. 17511301, Cytiva) under gentle rotation at room temperature for 2 hours. The resin was sequentially washed twice with 6.0 M guanidine hydrochloride/2.0 M NaCl and resuspended in 0.3 M guanidine hydrochloride. Reduction and alkylation steps were performed as follows: 10.0 mM dithiothreitol (DTT) treatment at room temperature for 1 hour, followed by 20.0 mM iodoacetamide incubation in darkness for 30 minutes. The resin was then washed twice with 50.0 mM NH4HCO3 solution and digested with trypsin (enzyme-to-protein ratio 1:30, w/w) at 37 °C for 12 hours. Finally, the enzymatic supernatant was collected by centrifugation, lyophilized, and stored. The proteomics experiment was performed in three independent biological replicates.", "projectTags" : [ ], "keywords" : [ "Amyloid", "Alzheimer's disease", "Photosensitizer", "Chemical proteomics", "Proximity labeling" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-18", "updatedDate" : "2026-03-17", "submissionDate" : "2026-03-17", "downloadCount" : 84, "avgDownloadsPerFile" : 6.0, "botCount" : 40, "hubCount" : 44, "organicCount" : 0, "percentile" : 4, "yearlyDownloads" : [ { "year" : "2026", "count" : 84 } ], "submitters" : [ "Jing Yan " ], "labPIs" : [ "Jing Yan" ], "affiliations" : [ "Research Center, The Second Affiliated Hospital of Dalian Medical University, Dalian, China" ], "instruments" : [ "timsTOF Pro" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "brain", "Mus musculus (Mouse)" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Brain" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "XB09826DA_hpc_X_Slot2-16_1_37323.d.rar", "XB09826DA_hpc_L_Slot2-15_1_37319.d.rar", "XB09826DA_cortex_L_Slot2-14_1_37313.d.rar", "XB09826DA_cortex_K_Slot2-14_1_37315.d.rar", "Results.rar", "XB09826DA_cortex_J_Slot2-14_1_37314.d.rar", "XB09826DA_hpc_V_Slot2-16_1_37321.d.rar", "checksum.txt", "XB09826DA_cortex_V_Slot2-13_1_37310.d.rar", "XB09826DA_hpc_J_Slot2-15_1_37317.d.rar", "XB09826DA_hpc_W_Slot2-16_1_37322.d.rar", "XB09826DA_cortex_W_Slot2-13_1_37311.d.rar", "XB09826DA_hpc_K_Slot2-15_1_37318.d.rar", "XB09826DA_cortex_X_Slot2-13_1_37309.d.rar" ], "highlights" : { } }, { "accession" : "PXD075718", "title" : "NicheProt: Cell-type-resolved proteomics of mouse spleen tissue compartments", "projectDescription" : "This dataset contains proteomic analyses that validate the spatial proteomic technology, NicheProt, and its application to investigate dendritic cell phenotypes in inflammed mouse spleens. NicheProt is a 3D optical microscopy-guided, photobleaching-mediated cell barcoding approach to isolate intact cell types from specific microanatomical niches. Integrating with sequential bottom-up LC-MS/MS analysis we identified two distinct CD11c⁺ dendritic cell phenotypes defined by their spatial distribution. These compartment-specific populations displayed differential expression of 54 proteins. This approach enables cell-type and microregion-resolved proteomic insights, revealing previously unrecognized cell subtypes and their roles within distinct tissue compartments.", "dataProcessingProtocol" : "Raw MS files were processed using label-free quantification (LFQ) and searched against a Swissprot mouse database (SP_mouse_2024_06; 17208 entries) using the Sequest HT node in Proteome Discoverer Software (version 3.0.0.757). Peptide and protein identifications are filtered at a 1% false discovery rate (FDR). Proteomics downstream data analysis and visualization were performed using R (version 2024.09.1+394).", "sampleProcessingProtocol" : "Spatially cell barcoded dendritic cells were dissociated from mouse spleen macrosections and isolated with fluorescence-activated cell sorting (FACS). The sorted cells were lysed at 95°C for 30 minutes followed by 65°C for 1 hour in lysis buffer (1× RIPA lysis buffer, 1× complete protease inhibitor, 1× PhosSTOP phosphatase inhibitor cocktail, and 3% (w/v) sodium dodecyl sulfate (SDS)). Extracted proteins were digested overnight on a S-trap column with Trypsin. LC-MS/MS analysis was conducted on a Vanquish Neo UHPLC equipped with a trap column (PepMap C18, 300 µm × 5 mm) and an IonOpticks Ultimate C18 column (1.7 µm; 75 µm × 25 cm) coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).", "projectTags" : [ ], "keywords" : [ "Cell barcoding", "Microscopy", "Dendritic cells", "Spatial proteomics", "Inflammation" ], "doi" : "10.6019/PXD075718", "submissionType" : "COMPLETE", "publicationDate" : "2026-03-19", "updatedDate" : "2026-03-17", "submissionDate" : "2026-03-17", "downloadCount" : 93, "avgDownloadsPerFile" : 7.1538463, "botCount" : 83, "hubCount" : 8, "organicCount" : 2, "percentile" : 5, "yearlyDownloads" : [ { "year" : "2026", "count" : 93 } ], "submitters" : [ "Steve Seung-Young Lee" ], "labPIs" : [ "Steve Seung-Young Lee" ], "affiliations" : [ "Department of Pharmaceutical Sciences, University of Illinois College of Pharmacy,Chicago,IL,USA Richard and Loan Hill Department of Biomedical Engineering, University of Illinois College of Engineering,Chicago, IL,USA University of Illinois Cancer Center,Chicago, IL,USA" ], "instruments" : [ "Orbitrap Exploris 480" ], "softwares" : [ ], "quantificationMethods" : [ "Absolute quantification" ], "sampleAttributes" : [ "Mus musculus (Mouse)", "dendritic cell" ], "organisms" : [ "Mus musculus", "Mus musculus (mouse)" ], "organismsPart" : [ "Spleen", "Dendritic cell" ], "diseases" : [ "Healthy", "Lipopolysaccharide-incuded inflammation" ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "Spleen Dendritic cell N/a Mus musculus Ac=ms:1001251;nt=trypsin Male Nt=carbamidomethyl;ac=unimod:4;ta=c;mt=fixed Lipopolysaccharide-incuded inflammation 1 Data-dependent acquisition 2 3 Nt=label free sample 4 Proteomic profiling by mass spectrometry 5 6 6-8 weeks Ac=ms:1003028; nt= orbitrap exploris 480 0.02 da Healthy 10 ppm Nt=oxidation;mt=variable;ta=m;ac=unimod:35 ; nt=acetylation;mt=variable;ac=unimod:1", "projectFileNames" : [ "CE3_2024Apr16_UIC_P24035_TCZ.raw", "CE3_2024May16_UIC_P24035.sdrf.tsv", "CE3_UIC_P24035_12TCZ3_AS1-9.mzML", "CE3_2024May16_UIC_P24035_AS5.raw", "CE3_UIC_P24035_12TCZ3_AS1-9.mzid", "CE3_2024May16_UIC_P24035_AS1.raw", "CE3_2024May16_UIC_P24035_AS3.raw", "checksum.txt", "CE3_2024Apr16_UIC_P24035_outside.raw", "CE3_2024Apr16_UIC_P24035_norm.raw", "CE3_2024May16_UIC_P24035_AS6.raw", "CE3_2024May16_UIC_P24035_AS4.raw", "CE3_2024May16_UIC_P24035_AS2.raw" ], "highlights" : { } }, { "accession" : "PXD075760", "title" : "An Lsr2-like xenogeneic silencer confers immunity against AT-rich bacteriophage infection", "projectDescription" : "Lsr2-like xenogeneic silencers are widespread nucleoid-associated proteins in Actinomycetota and are encoded by diverse bacteriophages. However, their potential roles during bacteriophage infection, one of the most direct encounters with foreign DNA, remain unexplored. In Corynebacterium glutamicum, the Lsr2-like protein CgpS is encoded within the mobile genetic element CGP3 and is known to silence transcription of foreign DNA within this region. Here we show that CgpS also restricts infection by the AT-rich bacteriophage Jean Grey. Upon phage DNA injection, CgpS binds phage-derived AT-rich sequences and represses early phage gene transcription. This transcriptional bottleneck prevents efficient phage genome replication and delays progression of the viral program. The resulting temporal window enables activation of host stress and defense pathways, including LexA-regulated SOS genes and additional antiviral systems. Our findings reveal a novel antiviral function for an Lsr2-like silencer and suggest that xenogeneic silencers can act as regulatory barriers that modulate the outcome of phage infection in Actinomycetota.", "dataProcessingProtocol" : "Samples were measured using the Vanquish Neo UHPLC System (ThermoFisher Scientific, Waltham, MA USA) coupled to an Orbitrap Exploris 480 mass spectrometer with a FAIMSpro interface. Peptides were separated on an µPAC NEO HPLC column (S/N 7501198, ThermoFisher Scientific, Waltham, MA USA) operated at 50°C, using 38-minute gradient (250 nl/min, 10% Buffer B at minute 4, 22% at minute 23, 45% at minute 31, 95% at min 34 until the end of the gradient). Eluting peptides were injected into the mass spectrometer at a static voltage of 2000 V, with a carrier gas flow of 3.6 L/min and an ion transfer tube temperature of 225 °C. FAIMS was operated at standard resolution and one compensation voltage of -45. MS1 scans were acquired at 120,000 resolution across a mass range of 400–1000 m/z, automatic maximum injection time, normalized AGC target of 3e6, RF Lens at 40%, using profile mode. Data independent MS/MS scans were acquired with an HRMS1 DIA method at 60,000 resolution, with an isolation window of 6 m/z (with loop N, 33 scans each), first mass at 200 m/z, HCD fragmentation at 30% normalized collision energy, AGC target of 1e6, and automatic maximum injection time. Raw data were analyzed using DIA-NN68 (v2.0.2, academia), searching against the custom databases. Search parameters included a step for FASTA digest for library-free search with deep learning-based spectra, RTs and Ims prediction, using Trypsin/P as protease, allowing for two missed cleavages and one variable modification. N-terminal methionine excision, cysteine carbamidomethylation, methionine oxidation, and N-terminal acetylation were included in the library, with peptide, precursor and fragment ranges set to default (6-36 amino acids, 1-4 charge states, 300-1800 precursor m/z range, 200-1800 fragment m/z range). Main search was performed with constructed library using MS1 quantification (--ms1-base-profile) using QuantUMS, at 1% precursor FDR with peptidoform scoring and FASTA protein names proteotypicity and otherwise default settings.", "sampleProcessingProtocol" : "Overnight cultures (n = 3 biological replicates) grown to stationary phase were used to inoculate 20 mL BHI medium to an initial OD₆₀₀ of 0.6. Cultures were incubated for 30 min at 30 °C with shaking at 110 rpm to allow metabolic reactivation. An initial sample (t₀) was collected prior to phage infection. For protein isolation, 2 mL culture aliquots were harvested by centrifugation at 13,000 rpm and 4 °C. Supernatants were carefully removed, and cell pellets were immediately frozen in liquid nitrogen. Phages were subsequently added at a multiplicity of infection (MOI) of 1, and cultures were incubated under the same conditions for 1 h. Samples were collected at 15-, 30-, and 60-min post infection following the same harvesting procedure. Cell pellets were resuspended in 300 µL proteomics lysis buffer (100 mM ammonium bicarbonate (ABC), 1% sodium lauryl sulfate (SLS), 1 mg/mL lysozyme) and incubated for 5 min at room temperature. The cell suspension was heat-treated at 95 °C for 10 min. Suspensions were then transferred to 2 mL FastPrep tubes (MP Biomedicals) containing 0.1 g of 0.1 mm glass beads (Scientific Industries Inc.) and cells were lysed by two cycles of bead beating using a FastPrep-25 5G (MP Biomedicals) at 6.0 m s⁻¹ for 60 s. The lysate was transferred to a new tube, and 600 µL of 100 mM ABC containing 10 U benzonase was added to each sample. Samples were mixed briefly by vortexing and incubated at 30 °C with shaking (350 rpm) for 1 h. Proteins were reduced by addition of tris(2-carboxyethyl)phosphine (TCEP) to a final concentration of 2 mM (40 °C, 10 min) and alkylated with iodoacetamide added to a final concentration of 4 mM (30 min, room temperature, in the dark). Samples were centrifuged at 10,000 × g for 10 min at 4 °C and the supernatant was transferred to a fresh tube. Proteins were precipitated by addition of five volumes of ice-cold acetone and incubation at −20 °C for 4 h. Proteins were collected by centrifugation at 10,000 × g for 10 min at 4 °C. The supernatant was discarded, and pellets were washed with 500 µL cold methanol, which was then removed by repeated precipitation. Dry protein pellets were resuspended in 200 µL of 100 mM ABC containing 0.1% SLS. Protein concentrations were determined using a Pierce™ BCA assay (Thermo Fisher Scientific). For digestion, 20 µg protein per sample was digested with 0.5 µg trypsin overnight at 30 °C. Following digestion, SLS was precipitated by addition of TFA to a final concentration of 1% (v/v), and the supernatant was collected. The supernatant was desalted for mass spectrometric analysis using C18 solid-phase columns (Chromabond C18 spin columns; Macherey-Nagel, Düren, Germany). The desalted peptides were used for LC–MS analysis.", "projectTags" : [ ], "keywords" : [ "Infection", "Corynebacterium glutamicum", "Proteomics", "Bacteriophage", "Dia" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-21", "updatedDate" : "2026-03-17", "submissionDate" : "2026-03-17", "downloadCount" : 9, "avgDownloadsPerFile" : 0.12328767, "botCount" : 0, "hubCount" : 9, "organicCount" : 0, "percentile" : 1, "yearlyDownloads" : [ { "year" : "2026", "count" : 9 } ], "submitters" : [ "Konstantinos Kalogeropoulos" ], "labPIs" : [ "Konstantinos Kalogeropoulos" ], "affiliations" : [ "Technical University of Denmark" ], "instruments" : [ "Orbitrap Exploris 480" ], "softwares" : [ ], "quantificationMethods" : [ "Relative quantification" ], "sampleAttributes" : [ "Corynebacterium glutamicum MB001" ], "organisms" : [ "Corynebacterium glutamicum mb001" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-independent acquisition" 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"20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP21.raw.quant", "20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP13.raw.quant", "report.log.txt", "20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP27.raw", "20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP14.raw.quant", "report.stats.tsv", "20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP22.raw", "20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP8.raw.quant", "20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP27.raw.quant", "CgluWT-PhageJG-combined.fasta", "20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP10.raw.quant", "20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP28.raw", "report-first-pass.stats.tsv", "20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP21.raw", "report.pg_matrix.tsv", "lib.parquet", "20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP17.raw.quant", "20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP16.raw", "20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP29.raw", "20251211_MH_ELBE_KK_FAIMS_1CV_uPAC50_38min_1475_DIA_NP20.raw.quant" ], "highlights" : { } }, { "accession" : "PXD075756", "title" : "An inflammasome-driven differentiation program in intestinal stem cells protects against Salmonella infection", "projectDescription" : "Intestinal stem cells (ISCs) are essential for sustaining epithelial renewal and barrier integrity, yet their role in orchestrating defense against enteric pathogens remains unclear. Here, we identify a stem cell–intrinsic immune mechanism whereby Lgr5⁺ ISCs detect intracellular Salmonella enterica and activate an inflammasome-dependent differentiation program. Using fluorescent-labeled Salmonella enterica, single-cell transcriptomics, fate mapping, organoid models, and genetic perturbations, we show that invaded ISCs undergo rapid reprogramming toward antimicrobial peptide-enriched Paneth cells via ASC (Pycard)-mediated inflammasome signaling. This fate switch enhances epithelial antimicrobial capacity and restricts pathogen persistence in the crypt. The response is Salmonella-specific and conserved in human intestinal organoids. Moreover, the invasion-associated transcriptional signature is enriched in ISCs from Crohn’s disease patients. Our findings reveal that ISCs act as active sensors of bacterial invasion and initiate epithelial remodeling through inflammasome signaling, highlighting stem cell plasticity as a frontline innate immune strategy.", "dataProcessingProtocol" : "Raw data was processed with DIA-NN v.2.2.0104. The data was searched in library-free mode against the M. musculus and S. Typhimurium Uniprot proteome databases appended with common lab protein contaminants and the following modifications: Carbamidomethylation of C as a fixed modification and oxidation of M and protein N-terminal acetylation as variable ones. Quantification was performed using the QuantUMS (high precision) algorithm. The Label-Free Quantification intensities were extracted and used for further calculations using Perseus v1.6.2.3105. Those intensities were log transformed and only proteins that had at least 3 valid values in at least one experimental group were kept. The remaining missing values were imputed (width=0.3 and down shift=1.8). A student’s t-test was performed to identify the proteins that are differentially expressed.", "sampleProcessingProtocol" : "Paneth cells (EpCAM+ CD24+ SSC-high) were FACS sorted and infected as described above. At different timepoints, Paneth cells were FACS sorted directly into 0.2% n-dodecyl β-D-maltoside (DDM) 150mM NaCl. The lysates were reduced with 6.25 mM dithiothreitol for 30 min followed by alkylation with 12.5 mM iodoacetamide in the dark for additional 30 min. Samples were then digested with 30ng of trypsin for 4 h at 37 °C. The digested peptides were acidified with 0.1% TFA. Samples were then vacuum-dried, resuspended in 0.1% TFA and kept at −80°C until analysis. Liquid chromatography Each sample was loaded using split-less nano-Ultra Performance Liquid Chromatography (nanoElute2, Bruker, Germany). The mobile phase was: A) H2O + 0.1% formic acid and B) acetonitrile + 0.1% formic acid. The peptides were separated using a trap Pepmap Neo C18 column (300μm ID x 5mm, Thermo scientific) and an Aurora column (75μm ID x 25cm, IonOpticks) at 0.3 µL/min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 2% to 35%B in 30 min, 35% to 95%B in 30 sec, maintained at 95% for 3 min and then back to initial conditions. Mass Spectrometry The nanoUPLC was coupled online through a 10um ZDV emitter to a timsTOF Ultra mass spectrometer (Bruker Daltonics, Germany). Data was acquired in parallel accumulation–serial fragmentation combined with data independent acquisition (DIA-PASEF) mode103. For ion mobility 1/K0 range was set to 0.70-1.30 V/cm2, ramp time of 166 msec and estimated cycle time of 1.20 sec. 27 windows of 25 Da with 1 Da overlap were set over a range of 335.3-1011.3 Da.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-05", "updatedDate" : "2026-03-17", "submissionDate" : "2026-03-17", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Vladyslav Holiar" ], "labPIs" : [ "Moshe Biton" ], "affiliations" : [ "Principal Investigator | The Ernst and Kaethe Ascher Career Development Chair | The Department of Immunology and Regenerative Biology | Weizmann-UK Building, Room 17 | Weizmann Institute of Science | Rehovot 76100, ISRAEL | Tel: +972-8-934-4937" ], "instruments" : [ "timsTOF Ultra" ], "softwares" : [ "Spectronaut" ], "quantificationMethods" : [ "label-free quantification" ], "sampleAttributes" : [ "bacterial infectious disease", "small intestine", "Mus musculus (Mouse)", "paneth cell of epithelium of small intestine" ], "organisms" : [ "Mus musculus", "Mus musculus (mouse)" ], "organismsPart" : [ "Paneth cell of epithelium of small intestine", "Small intestine" ], "diseases" : [ "Bacterial infectious disease", "Uninfected", "Infected with s. typhimurium" ], "references" : [ ], "experimentTypes" : [ "Top-down proteomics", "Data-independent acquisition" ], "sdrf" : "Uninfected Mus musculus Trypsin Label free Paneth cells Not available years Timstof ultra Infected with s. typhimurium Small intestine", "projectFileNames" : [ "TTU_MB_24932_24-4_21092025_9-23-2025_6453.d.zip", "MBI_24932_all_groups_3vv_in_each_group_protein_report.xlsx", "TTU_MB_24932_24-C2_21092025_9-23-2025_6455.d.zip", "TTU_MB_24932_12-2_21092025_9-24-2025_6467.d.zip", "TTU_MB_24932_24-C1_21092025_9-23-2025_6458.d.zip", "TTU_MB_24932_0-1_21092025_9-24-2025_6462.d.zip", "TTU_MB_24932_0-4_21092025_9-24-2025_6472.d.zip", "TTU_MB_24932_24-3_21092025_9-23-2025_6456.d.zip", "TTU_MB_24932_24-1_21092025_9-23-2025_6454.d.zip", "TTU_MB_24932_2-1_21092025_9-24-2025_6468.d.zip", "TTU_MB_24932_12-4_21092025_9-24-2025_6471.d.zip", "TTU_MB_24932_24-C3_21092025_9-23-2025_6457.d.zip", "TTU_MB_24932_2-3_21092025_9-24-2025_6470.d.zip", "checksum.txt", "TTU_MB_24932_0-3_21092025_9-24-2025_6469.d.zip", "TTU_MB_24932_24-C4_21092025_9-23-2025_6460.d.zip", "TTU_MB_24932_2-4_21092025_9-24-2025_6465.d.zip", "TTU_MB_24932_12-1_21092025_9-24-2025_6466.d.zip", "TTU_MB_24932_2-2_21092025_9-24-2025_6461.d.zip", "TTU_MB_24932_0-2_21092025_9-24-2025_6463.d.zip", "TTU_MB_24932_12-3_21092025_9-24-2025_6464.d.zip", "sdrf.tsv", "TTU_MB_24932_24-2_21092025_9-23-2025_6459.d.zip" ], "highlights" : { } }, { "accession" : "PXD075707", "title" : "A Chemoproteomic Atlas of the Human Purine Interactome for Regioselective Ligand Discovery", "projectDescription" : "Purines are essential bioactive molecules that interact with a large fraction of the human proteome. Despite their importance, the scope of actionable purine-binding pockets for ligand discovery remains limited. Here, we developed a quantitative chemoproteomics platform using sulfonyl-purine (SuPUR) chemistry to produce a massive and functional map of the human purine interactome. The SuPUR platform captured 31,000+ targetable tyrosine and lysine sites, representing the most comprehensive beyond cysteine chemoproteomics database for enabling protein ligand discovery. SuPUR ligands that bind through a regioselective fashion serve as enabling starting points for developing potent (nanomolar) and proteome-wide-selective modulators of enzymatic and protein-protein interaction function. Phenotypic screening identified a site-specific (Y237) and regioselective SuPUR ligand of ACAT2 to reveal an unexpected metabolic dependency in cancer cells. A crystal structure of SuPUR ligand-bound ACAT2 revealed the purine group binds deep in the CoA pocket forming key interactions with catalytic residues via a water bridge to guide future structure-based ligand design.", "dataProcessingProtocol" : "LC-MS/MS evaluation of TMT-modified peptides was performed with a Vanquish Neo UHPLC coupled to an Orbitrap Exploris 480 mass spectrometer. Peptides were separated on an 140 min gradient by reverse phase LC using 3 µm C18 (20 cm) as follows: (A: 0.1% formic acid in H2O; B: 80% ACN, 0.1% formic acid in H2O: 0–2 min 4% B, 450 nL/min; 2–2.5 min 5% B, 300 nL/min; 2.5–78 min 25% B, 300 nL/min; 78–123 45% B, 300 nL/min; 123–124.973 min 99% B, 450 nL/min; 124.973-127.604 min 99% B, 450 nL/min; 127.604-129.577 min 1% B, 450 nL/min; 129.577-140 min 1% B, 450 nL/min). Data was acquired using a top 30 ddMS2 method. MS1 spectra were acquired at 120K resolution with a max IT of 20 ms and MS2 spectra were taken with an AGC of 25K, quadrupole isolation width of 0.7 m/z, and max IT of 50 ms. Data analysis Peptide identification and quantification for TMT-tagged peptides was performed using Proteome Discoverer 3.0 housing a PMI-Byonic node. MS2 spectra were searched against the human protein database (UniProt, download date 01/17/2024) using the following parameters: ≤ 2 missed cleavages, 10 ppm precursor mass tolerance, 20 ppm fragment mass tolerance, and 1% protein false discovery rate. Modifications considered included methionine oxidation (+15.9949, M, variable), cysteine carbamidomethylation (+57.021464, C, fixed), and TMT modification (+229.1629, N-term, K, variable). Peptides used for protein quantification met the following quality control criteria: PMI-Byonic Score ≥300, delta ppm err. 5, co-isolation interference threshold ≤ 50%, reporter ion S/N ≥10, TMT-modified, and were present in n = 2 biological replicates. Proteins were quantified had 3 or more unique, quality peptides matched. TMT channels were normalized to total peptide amount. Volcano plots were generated based on protein abundance ratios (inhibitor/vehicle) with cutoffs of Log2 FC ≥ 0.5, and p-value ≤ 0.05.", "sampleProcessingProtocol" : "Tandem mass tag (TMT) labeling 32 The peptide concentration was determined using Pierce Quantitative Colorimetric Peptide Kit. Peptides were normalized to a concentration of 5 µg/µL in 200 Mm EPPS (pH 8.5). For TMT labeling, 10 µL of peptides were mixed with 5 µL of TMTsixplex isotopomers reagent (100 µg) in PCR tubes, and the reaction was incubated for 1 h at RT with shaking at 500 rpm. The reaction was quenched by adding 1.0 µL of 5% hydroxylamine to each tube, followed by incubation at RT for 15 min with shaking at 500 rpm. Subsequently, 1 µL of formic acid (FA) was added to each tube to acidify and neutralize the excess hydroxylamine. All TMT-labeled samples were combined into one low-binding tube and dried using SpeedVac. Enrichment of SuPUR probe modified peptides Samples were resuspended in 300 µL Optima water, and 5 µL of resuspension was removed for non-enriched proteomics analysis (The same LC-MS/MS method used for post-enrichment competitive TMT-ABPP was applied). The resulting samples (295 µL) were incubated with 100 µL of avidin agarose and diluted with DPBS to a final volume of 5.5 mL. The mixture was incubated at RT for 1 h, followed by centrifugation at 1,400 × g for 1 minute to pellet the beads. The supernatant was then carefully decanted. The beads were washed three times with 10 mL of AmBic and an additional three times with 10 mL of Optima-grade water. Probe-modified peptides were eluted by incubating the beads three times with elution buffer (50% acetonitrile, 0.1% formic acid). Eluted samples were snap-frozen and subsequently dried using SpeedVac. Dried samples were resuspended in 50 µL of mobile phase A (0.1% FA in Optima water). Peptide cleanup was performed using StageTips (Octadecyl C18 47 mM, Millipore Sigma, Cat#173427). The peptides were snap-frozen and dried using SpeedVac. Samples were then resuspended in 25 µL of mobile phase A and prepared for LC-MS/MS analysis.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "10.6019/PXD075707", "submissionType" : "COMPLETE", "publicationDate" : "2026-03-29", "updatedDate" : "2026-03-16", "submissionDate" : "2026-03-16", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Ku-Lung Hsu" ], "labPIs" : [ "Ku-Lung Hsu" ], "affiliations" : [ "Department of Chemistry, University of Texas at Austin, Austin" ], "instruments" : [ "Q Exactive" ], "softwares" : [ ], "quantificationMethods" : [ "TMT" ], "sampleAttributes" : [ "prostate adenocarcinoma", "embryonic stem cell", "Homo sapiens (Human)", "squamous epithelial cell" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Squamous epithelial cell", "Embryonic stem cell" ], "diseases" : [ "Prostate adenocarcinoma" ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "20260109_BoDo_SV01_ZH_6_199_TMT1_T1_2a_c.raw", "20260109_BoDo_SV01_ZH_6_199_TMT2_T1_2a_c.raw", "20241130_BoDo_OM05_ZH_5_065_TMT_T2_S6e.raw", "20250102_BoDo_ZH03__ZH_5_091_TMT_LC_S7d.raw", "20240412_VaPa_XJ07_ZH_3_035_TMT_LC_1_HEK_IN_SITU_5d.raw", "20250129_BoDo_ZH03_ZH_5_099_TMT1_T1_UE_S6h.raw", "checksum.txt", "20241130_BoDo_OM05_ZH_5_065_TMT2_S6e.raw", "ABHD10_3BPs_S_and_M.mzML", "20240412_VaPa_XJ07_ZH_3_035_TMT_1_HEK_IN_SITU_sol.mzML", "20250110_BoDo_ZH03_ZH_5_099_TMT1_T2_5h.raw", "20240613_BoDo_MLH01_ZH_3_081_TMT2_T1_rep2_128_5_um_and_129_2_um_4h.raw", "20240412_VaPa_XJ07_ZH_3_035_TMT_1_HEK_IN_SITU_sol.mzid", "ABHD10_3BPs_S_and_M.mzid", "20240423_VaPa_XJ07_ZH_3_047_TMT_LC_1_HEK_IV_S2b.raw", "20240619_BoDo_MLH01_ZH_3_097_TMT2_LC_S5e.raw", "20241128_BoDo_OM05_ZH_5_065_TMT_S6e.raw", "20240619_BoDo_MLH01_ZH_3_097_TMT2_T2_rep3_4h.raw", "20240503_VaPa_XJ07_ZH_3_055_TMT1_LC_2_5d.raw", "20241030_ZH_BoDo_MLH01_ZH_5_029_TMT1_T2_3n.raw", "20250110_BoDo_ZH03_ZH_5_099_TMT2_T2_S6i.raw", "20240216_BoDo_MLH01_LF_ZH_3_015_MEM_FP_4f.raw", "20240628_BoDo_MLH01_ZH_3_103_TMT2_T1_rep2_and_3_3f.raw", "20250318-BoDo_ZH03_ZH_5_143_TMT1_T1_3g.raw", "20240619_BoDo_MLH01_ZH_3_097_TMT1_LC_S5e.raw", "20240412_VaPa_XJ07_ZH_3_035_TMT_1_HEK_IN_SITU_REP1_4g.raw", "20240619_BoDo_MLH01_ZH_3_097_TMT3_T2_rep1_4h.raw", "20240216_BoDo_MLH01_LF_ZH_3_021_SOL_ABHD10_FP_4f.raw", "20241216_BoDo_OM05_ZH_5_081_TMT1_6d.raw", "20241130_BoDo_OM05_ZH_5_065_TMT_UE_BP2_S6f.raw", "20240216_BoDo_MLH01_LF_ZH_3_021_MEM_ABHD10_FP_4f.raw", "ZH_1_049_2_FP_MIX.mzML", "ZH_1_049_2_FP_MIX.mzid", "20250129_BoDo_ZH03_ZH_5_099_TMT1_T2_UE_S6h.raw", "20240619_BoDo_MLH01_ZH_2-097_3reps_final.mzML", "ZH_1_049_2_FP_MIX.raw", "20250129_BoDo_ZH03_ZH_5_099_TMT2_T1_UE_S6j.raw", "20241030_ZH_BoDo_MLH01_ZH_5_029_TMT2_T1_3n.raw", "20240628_BoDo_MLH01_ZH_3_112_TMT1_LC_5d.raw", "20240619_BoDo_MLH01_ZH_2-097_3reps_final.mzid", "20241030_ZH_BoDo_MLH01_ZH_5_029_TMT2_T2_3n.raw", "20240216_BoDo_MLH01_LF_ZH_3_015_SOL_FP_4f.raw", "20250102_BoDo_ZH03__ZH_5_091_TMT_ACAT2.mzML", "ZH-1-049-2_SOL.mzid", "20241216_BoDo_OM05_ZH_5_081_TMT.mzML", "20240216_BoDo_MLH01_LF_ZH_3_021_SOL_ABHD10_REP2_FP_4f.raw", "20250102_BoDo_ZH03__ZH_5_091_TMT_ACAT2.mzid", "ZH-1-049-2_SOL.mzML", "20240613_BoDo_MLH01_ZH_3_081_TMT1_LC_S5e.raw", "20240503_VaPa_XJ07_ZH_3_055_TMT_1_HEK_TMT1_IS_ABHD10_rep2_4g.raw", "20250318-BoDo_ZH03_ZH_5_143_TMT2_UE1_S2e.raw", "20240628_BoDo_MLH01_ZH_3_103TMT.mzML", "20250110_BoDo_ZH03_ZH_5_099_TMT1_T2.mzid", "20250110_BoDo_ZH03_ZH_5_099_TMT2_T1_S6i.raw", "20250318-BoDo_ZH03_ZH_5_143_TMT2_T1_3h.raw", "20240628_BoDo_MLH01_ZH_3_103TMT.mzid", "20241216_BoDo_OM05_ZH_5_081_TMT.mzid", "20250206_BoDo_ZH03_ZH_5_099_TMT1_5h.raw", "20260109_BoDo_SV01_ZH_6_199_TMT1_T2_2a_c.raw", "20250110_BoDo_ZH03_ZH_5_099_TMT1_T2.mzML", "20241128_BoDo_OM05_ZH_5_065_TMT_UE_S6f.raw", "20240216_BoDo_MLH01_LF_ZH_3_021_MEM_ABHD10_REP2_FP_4f.raw", "20250318-BoDo_ZH03_ZH_5_143_TMT1_UE1_S2d.raw", "20250102_BoDo_ZH03__ZH_5_091_TMT_S7c.raw", "20260109_BoDo_SV01_ZH_6_199_TMT2_T2_2a_c.raw", "20240423_VaPa_XJ07_ZH_3_047_TMT_1_HEK_IV_rep1_and_2_3e.raw", "20250318-BoDo_ZH03_ZH_5_143_TMT_3BRs.mzML", "20240628_BoDo_MLH01_ZH_3_112_TMT2_T1_REP3_4g.raw", "20250129_BoDo_ZH03_ZH_5_099_TMT2_T2_UE_S6j.raw", "20240628_BoDo_MLH01_ZH_3_103_TMT1_LC_S2c.raw", "20250318-BoDo_ZH03_ZH_5_143_TMT_3BRs.mzid", "20241216_BoDo_OM05_ZH_5_081_TMT1_UE_S7d.raw" ], "highlights" : { } }, { "accession" : "PXD075691", "title" : "Stress-Specific Carbonylation and Proteasome 20S Activity in Potato under Drought, Elevated Temperature, and Combined Stresses: Linking Oxidative Damage to Proteome Regulation", "projectDescription" : "This study investigates stress-specific patterns of protein carbonylation and proteome re-modelling in potato leaves subjected to drought, elevated temperature, and their combina-tion. The research was conducted at the Department of Potato Agronomy of the Plant Breeding and Acclimatization Institute – National Research Institute. The Polish potato cultivar Solanum tuberosum L. ‘Lech’, known for its sensitivity to soil drought, was used in the study.Two weeks after tuber initiation (approximately BBCH 40), plants were subjected to four experimental treatments for a duration of 14 days: Control (C): maintained optimal hydration (70% field water capacity, FWC) and standard temperature conditions (20/16°C day/night). Drought (D): water withheld to reach and maintain 40% FWC at 20/16°C. Elevated Temperature (HT): plants exposed to 38/25°C (day/night) under optimal hydration (70% FWC). Combined Stress (D+HT): plants exposed to 38/25°C (day/night) with water withheld to 40% FWC.", "dataProcessingProtocol" : "Differential spots were excised, destained, and subjected to in-gel trypsin digestion. Peptides were analyzed by LC–MS/MS using an Orbitrap Exploris 480 (Thermo Fisher Scientific). MS/MS spectra were searched against the Solanum tuberosum NCBI database using the MASCOT engine. Search parameters included: ±5 ppm peptide tolerance, ±0.01 Da fragment tolerance, and two missed cleavages. Carbamidomethylation was set as a fixed modification, while methionine oxidation was considered a variable modification.", "sampleProcessingProtocol" : "Protein carbonylation was determined according to Levine et al. (1994) [41] using DNPH (2,4-dinitrophenylhydrazine) derivatization. The absorbance was measured at 375 nm, and results were expressed as nmol carbonyl per mg of protein. The 20S proteasome activity was measured fluorometrically using 5 µg of total protein with a commercial as-say kit (MAK172, Sigma-Aldrich), following the manufacturer’s instructions. Total Protein Extraction. Leaf tissue (150 mg) was ground in liquid nitrogen, and proteins were precipitated with ice-cold 10% TCA/acetone. The resulting pellet washed sequentially with 10% TCA/acetone, 80% methanol containing 0.1 M ammonium acetate, and 80% acetone. The dried pellet was resuspended in a mixture of Tris-buffered phenol (pH 8.0) and SDS extraction buffer (0.1 M Tris-HCl, 30% sucrose, 5% β-mercaptoethanol, 2% SDS). After centrifugation, the phenolic phase was collected. Proteins were precipitated overnight with 0.1 M ammonium acetate in 80% methanol at −20 °C, washed with meth-anol and acetone, and finally dissolved in IEF buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT). Protein concentration was determined using the Bradford-based method adapted for proteomic samples [42]. Two-Dimensional Gel Electrophoresis (2D-PAGE) and Immunoblotting. For proteo-me mapping, 120 µg of protein was loaded onto 7-cm pH 4–7 NL Immobiline DryStrips. Following isoelectric focusing (IEF), strips were equilibrated in buffers containing DTT (reduction) and iodoacetamide (alkylation) and separated on 11% SDS-PAGE gels. Gels were stained with colloidal Coomassie Brilliant Blue G-250. For carbonylation detection, 20 µg of protein was derivatized with DNPH before IEF. After 2D separation, proteins were electro-transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk and incubated with primary anti-DNP antibodies (Sigma-Aldrich), followed by alkaline phosphatase-conjugated secondary antibodies. Signals were visualized using NBT/BCIP substrate and the reaction was terminated with distilled water. Four biological replicates were conducted for each experimental variant, including protein purification, two-dimensional electrophoresis (2DE), membrane transfer, and immunochemical staining (n = 4). Gels and PVDF membranes were scanned using an Image Scanner III (GE Healthcare), and their digital images were analyzed with Delta2D Version 2.0 software (DECODON GmbH, Greifswald, Germany). Differential proteins were identified by comparing plants subjected to drought, high temperature, and a combination of these stresses with control leaves from the same cul-ti-var. After correcting for positional spot variations, a virtual fused image was created to consolidate the information from all images into one composite image, followed by the detection of a consensus spot pattern. For spot quantification based on size and intensity, a standard procedure embedded in the software was applied to all membrane images from the experiment. Normalized spot intensities were calculated by relating the intensity of each in-di-vidual spot to the total intensity of all detected spots in the gel or membrane image. The accuracy of gel or membrane identity between biological replicates was assessed using principal component analysis (PCA), which served as both a quality control step and a means of comparing all experimental variants. The selection of differential proteins was based on mean spot intensity and was evaluated using one-way ANOVA with an ad-just-ed Bonferroni correction (critical p-value < 0.01). Images of selected spots on mem-branes were overlaid onto the corresponding gel images, and the selected spots were then excised from the gels for identification purposes.", "projectTags" : [ ], "keywords" : [ "Solanum tuberosum l.", "Protein carbonylation", "Elevated temperature", "Drought stress" ], "doi" : "10.6019/PXD075691", "submissionType" : "COMPLETE", "publicationDate" : "2026-04-05", "updatedDate" : "2026-03-16", "submissionDate" : "2026-03-16", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Marta Gietler" ], "labPIs" : [ "Marta Gietler" ], "affiliations" : [ "Department of Biochemistry and Microbiology, Institute of Biology, Warsaw University of Life Scienc-es-SGGW, Poland" ], "instruments" : [ "Orbitrap Fusion" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "leaf", "Solanum tuberosum (Potato)" ], "organisms" : [ "Solanum tuberosum (potato)" ], "organismsPart" : [ "Leaf" ], "diseases" : [ ], "references" : [ "Boguszewska-Mańkowska D, Fidler-Jarkowska J, Gietler M, Nykiel M. Stress-Specific Carbonylation and Proteasome 20S Activity in Potato Under Drought, Elevated Temperature, and Combined Stresses: Linking Oxidative Damage to Proteome Regulation. 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In budding yeast, cells are born with a similar amount of the G1/S inhibitor protein Whi5 that is then diluted by growth throughout G1. As cells grow, Whi5 concentration decreases and cells become more likely to enter the cell cycle. Cells are born in G1 with similar amounts of Whi5 because of the size-independent (sub-scaling) expression of WHI5 mRNA during S/G2/M phases and the equal partitioning of Whi5 protein at division. While the latter is known to be achieved by association with chromatin before anaphase, the mechanism for the former is poorly understood. Through systematic mutations of the WHI5 promoter, we discovered that WHI5’s core promoter region located -126 to -75 base pairs upstream of the start codon is responsible for sub-scaling expression. This sequence contains a repeating array of binding sites for the transcription factors Fkh1 and Fkh2. Mutation of any of these sites, deletion of either FKH1 or FKH2, or preventing Fkh1 or Fkh2 dimerization weakens the sub-scaling of WHI5 transcription. Taken together with structural predictions and a mathematical model of cooperative Fkh-DNA binding, we conclude that WHI5’s sub-scaling transcription is regulated by a Fkh1/2 heteropolymer binding an array of sites in its core promoter.", "dataProcessingProtocol" : "All raw files were searched using the Andromeda engine embedded in MaxQuant (v2). Reporter ion MS3 search was conducted using TMTpro (16-plex) isobaric labels. Variable modifications included oxidation (M) and protein N-terminal acetylation. Carbamidomthyl (C) was a fixed modification. The number of modifications per peptide was capped at five. Digestion was set to tryptic (proline-blocked). Database search was conducted using the UniProt proteome - Ecoli_UP000000625_83333. The minimum peptide length was 7 amino acids. 1% FDR was determined using a reverse decoy proteome.", "sampleProcessingProtocol" : "The pellets were then resuspended in a denaturation/reduction buffer (0.07M Tris-Cl, 5% (v/v) mercaptoethanol, 0.6% (w/v) SDS and 15% (v/v) glycerol) by boiling for 10 min at 95 °C with intermittent vortexing. Debris was pelleted by centrifugation for 10 minutes (10,000 ×g). Cleared supernatants were alkylated with 5mM iodoacetamide, and then precipitated with three volumes of a solution containing 50% acetone and 50% ethanol. Proteins were re-solubilized in 2 M urea, 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl, and then digested with TPCK-treated trypsin (50:1) overnight at 37 °C. Trifluoroacetic acid and formic acid were added to the digested peptides for a final concentration of 0.2%. Peptides were desalted with a Sep-Pak 50mg C18 column (Waters). The C18 column was conditioned with 500 µL of 80% acetonitrile and 0.1% acetic acid and then washed with 1000 µL of 0.1% trifluoroacetic acid. After samples were loaded, the column was washed with 2000 µL of 0.1% acetic acid followed by elution with 400 µL of 80% acetonitrile and 0.1% acetic acid. The elution was dried in a Concentrator at 45 °C. De-salted peptides were resuspended in 0.1% Formic acid. For each sample, 25 µg of desalted peptide samples were resuspended in 20 µL of 100 mM Triethylammonium bicarbonate solution and labeled with 16-plex TMTpro at a ratio 4:1 (TMT:peptide). Total reaction volume was less than 25 µL. The labeling reaction was quenched with a final concentration of 0.5% hydroxylamine for 15 min. Labeled peptides were pooled and acidified to pH ∼ 2 using drops of 10% TFA. Excess TMT label was removed by re-running the pooled sample through a Sep-Pak 50-mg C18 column (as described above). TMT-labeled peptides were resuspended in 0.1% formic acid analyzed on a Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with a Thermo EASY-nLC 1200 LC system (Thermo Fisher Scientific, San Jose, CA). Peptides were separated by capillary reverse phase chromatography on a 25 cm column (75 µm inner diameter, packed with 1.6 µm C18 resin, AUR2-25075C18A, Ionopticks, Victoria Australia). Peptides were introduced into the Fusion Lumos mass spectrometer using a 180-min stepped linear gradient at a flow rate of 300 nL / min. The steps of the gradient are as follows: 6–33% buffer B (0.1% (v:v) formic acid in 80% acetonitrile) for 145 min, 33-45% buffer B for 15min, 40–95% buffer B for 5 min and maintain at 90% buffer B for 5 min. The column temperature was maintained at 50°C throughout the procedure. Xcalibur software (v.4.4.16.14) was used for the data acquisition and the instrument was operated in data-dependent mode. Advanced peak detection was disabled. Survey scans were acquired in the Orbitrap mass analyzer (centroid mode) over the range 380–1,400 m/z with a mass resolution of 120,000 (at m/z 200). For MS1, the normalized AGC target (%) was set at 250 and maximum injection time was set to 100 ms. Selected ions were fragmented by collision-induced dissociation (CID) with normalized collision energies of 34 and the tandem mass spectra were acquired in the ion trap mass analyzer with the scan rate set to ‘Rapid’. The isolation window was set to the 0.7 m/z window. For MS2, the normalized AGC target (%) was set to ‘Standard’ and maximum injection time to 35 ms. Repeated sequencing of peptides was kept to a minimum by dynamic exclusion of the sequenced peptides for 30 s. The maximum duty cycle length was set to 3 s. Relative changes in peptide concentration were determined at the MS3 level by isolating and fragmenting the five most dominant MS2 ion peaks.", "projectTags" : [ ], "keywords" : [ "Whi5", "Size scaling", "Cell size" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-31", "updatedDate" : "2026-03-14", "submissionDate" : "2026-03-14", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Michael Lanz" ], "labPIs" : [ "Jan Skotheim" ], "affiliations" : [ "Department of Biology, Stanford University" ], "instruments" : [ "Orbitrap Fusion Lumos" ], "softwares" : [ ], "quantificationMethods" : [ "TMT" ], "sampleAttributes" : [ "Saccharomyces cerevisiae (Baker's yeast)" ], "organisms" : [ "Saccharomyces cerevisiae (baker's yeast)" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "ForkHeadCellSize_Std_F13_R1_T1_MS3TMT001_001357_JASK011_FL2.raw", "ForkHeadCellSize_Std_F5_R1_T1_MS3TMT001_001348_JASK011_FL2.raw", "ForkHeadCellSize_Std_F17_R1_T1_MS3TMT001_001361_JASK011_FL2.raw", "ForkHeadCellSize_Std_F1_R1_T3_MS3TMT001_001344_JASK011_FL2.raw", "ForkHeadCellSize_Std_F9_R1_T1_MS3TMT001_001353_JASK011_FL2.raw", "ForkHeadCellSize_Std_F12_R1_T1_MS3TMT001_001356_JASK011_FL2.raw", "ForkHeadCellSize_Std_F2_R1_T1_MS3TMT001_001345_JASK011_FL2.raw", "ForkHeadCellSize_Std_F16_R1_T1_MS3TMT001_001360_JASK011_FL2.raw", "ForkHeadCellSize_Std_F6_R1_T1_MS3TMT001_001349_JASK011_FL2.raw", "checksum.txt", "ForkHeadCellSize_Std_F15_R1_T1_MS3TMT001_001359_JASK011_FL2.raw", "ForkHeadCellSize_Std_F11_R1_T1_MS3TMT001_001355_JASK011_FL2.raw", "ForkHeadCellSize_Std_F3_R1_T1_MS3TMT001_001346_JASK011_FL2.raw", "ForkHeadCellSize_Std_F1_R1_T1_MS3TMT001_001342_JASK011_FL2.raw", "msms.txt", "ForkHeadCellSize_Std_F8_R1_T1_MS3TMT001_001351_JASK011_FL2.raw", "ForkHeadCellSize_Std_F14_R1_T1_MS3TMT001_001358_JASK011_FL2.raw", "ForkHeadCellSize_Std_F10_R1_T1_MS3TMT001_001354_JASK011_FL2.raw", "ForkHeadCellSize_Std_F4_R1_T1_MS3TMT001_001347_JASK011_FL2.raw", "ForkHeadCellSize_Std_F18_R1_T1_MS3TMT001_001362_JASK011_FL2.raw", "ForkHeadCellSize_Std_F7_R1_T1_MS3TMT001_001350_JASK011_FL2.raw", "ForkHeadCellSize_Std_F1_R1_T2_MS3TMT001_001343_JASK011_FL2.raw" ], "highlights" : { } }, { "accession" : "PXD075602", "title" : "Probing molecular diversity of brain cells with chemically modified aptamers", "projectDescription" : "We developed a volumetric correlated light and electron microscopy (CLEM) approach that uses slow off-rate modified aptamers (SOMAmers) for fluorescence labeling of cells in ultrastructurally reconstructable electron micrographs. The small size of aptamers enables intracellular labeling in fixed brain tissue without detergent permeabilization, thereby preserving tissue ultrastructure and allowing high-resolution electron microscopy imaging and accurate cellular segmentation. This method enables labeling of a wide range of molecular biomarkers, providing a powerful strategy to integrate molecular information with volumetric electron microscopy datasets. As a result, it facilitates molecularly annotated ultrastructural studies of complex tissues, including investigations of neural circuits. To ensure that the aptamers used in this method bind specifically to their intended protein targets, we validated target binding using pull-down mass spectrometry. These results were further confirmed through complementary approaches including genetic correlation analyses, proximity extension assays, and comparisons with commercial antibody labeling.", "dataProcessingProtocol" : "MS spectral analysis was performed using an MSE method on a Waters Xevo G2-S QTof mass spectrometer. The peptides were ionized using a narrow-bore ESI probe in positive ionization mode. A mass range of 50–1200 m/z (continuum mode) was utilized with a scan rate of 0.5 s and low/high collision energies of 4 V and 15-40 V, respectively. The capillary voltage was set to 1 kV, and the cone voltage was 40 V, with a cone gas flow of 40 L/h and a desolvation gas flow of 400 L/h (400°C). The source temperature was maintained at 120°C. Results from LC-MS/MS were analyzed and compared against protein databases using ProteinLynx Global Server v3.0.3 (PLGS).", "sampleProcessingProtocol" : "SOMAmer reagents (40 pmol) containing a terminal photocleavable biotin were immobilized on streptavidin-coated beads and incubated with 50 pmol recombinant protein target for 3 hours at 28°C in 190 µL Selection Buffer containing 20 µM polyanionic competitor (Z-Block26). SOMAmer-protein complexes were then challenged with 10 mM dextran sulfate (m.w. 5,000) and washed 6 times with 190 µL Selection Buffer. Samples were irradiated with a UV Bench Lamp XX-15L (15 W, 365 nm; Analytik Jena) for 17 min at 25°C to release SOMAmer-protein complexes via the photocleavable linker. SOMAmer-protein complexes were digested with 1 nM trypsin in 8 M urea, 200 mM iodoacetamide, 200 mM dithiothreitol, and 25 mM ammonium bicarbonate. Peptide fragments were desalted using an OASIS Prime HLB plate (Waters, cat. #186008053), eluted with 70% acetonitrile, dried, and reconstituted in 15 µL of 2% acetonitrile/0.1% formic acid. UPLC was performed on a Waters ACQUITY M-Class UPLC. Analytes were separated on a nanoEase HSS T3 100 Å, 1.8 µm, 300 µm × 150 mm column (Waters, cat. #186009249) with a 5 µL injection volume and 5 µL/min flow rate. The gradient consisted of 3-27% buffer B (0.1% formic acid in acetonitrile) in buffer A (0.1% formic acid in H₂O) from 0-13 min, followed by 27-95% buffer B from 13-14 min. The trap column and LC were both maintained at 60°C.", "projectTags" : [ ], "keywords" : [ "Pulldown ms", "Neuroscience" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-17", "updatedDate" : "2026-03-13", "submissionDate" : "2026-03-13", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Xiaotang Lu" ], "labPIs" : [ "Xiaotang Lu" ], "affiliations" : [ "University of Illinois Urbana-Champaign" ], "instruments" : [ "Q-Tof Ultima" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "brain", "Mus musculus (Mouse)" ], "organisms" : [ "Homo sapiens (human)", "Mus musculus (mouse)" ], "organismsPart" : [ "Brain" ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.1038/S41467-026-72180-7" ], "experimentTypes" : [ "Affinity purification coupled with mass spectrometry proteomics", "MSE" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "NPTXR_15511-37.raw.zip", "identification.csv", "NPY_20590-13.raw.zip", "ELAV2_2173-25.raw.zip", "DHPR_11257-1.raw.zip", "MAG_20579-50.raw.zip", "Peroxiredoxin-6_5018-68.raw.zip", "GFP_3849-56.raw.zip", "PENK_9076-25.raw.zip", "PKC-G_5476-66.raw.zip" ], "highlights" : { } }, { "accession" : "PXD075538", "title" : "Deciphering NAC53, NAC78 interactome in response to Pst infection; NAC53, NAC78, MDP25 subcellular interactomes and identifying ubiquitination sites of NAC53 and NAC78 upon HRD1 E3 ligase mediated ubiquitination.", "projectDescription" : "Analysis of NAC53 and NAC78 interactome in normal condition or upon Pseudomonas syringea pv. tomato DC3000 ∆HopQ infection. Constructs promUBQ10::eGFP-NAC53/eGFP-NAC78 are used as baits and promUBQ10::eGFP is used as control. Analysis of ubiquitination sites on NAC53 and NAC78 upon HRD1a/b ubiquitination. Analysis of NAC53, NAC78 and MDP25 subcellular interactome. For NAC53 and NAC78, dual tagged constructs pLjUBQ1::mCerrulean-NAC53/78-mCherry were used to pull-down N-ter moieties (cytosolic/nuclear localization) or C-ter moieties (endoplasmic reticulum localization). pUBQ10::eGFP and p35S::mCherry-HDEL are used as controls. For MDP25, protein variants were used, MDP25FL (plasma membrane localization), MDP25∆N25 (cytosolic localization) or N25MDP25 (plasma membrane localization). pUBQ10::eGFP is used as control.", "dataProcessingProtocol" : "Acquired MS spectra were processed with MaxQuant software package version 1.5.2.8 with an integrated Andromeda search engine. For in vivo IP samples, database search was performed against a N. benthamiana database, the sequences of eGFP-NAC53/78, mCerrulean3-NAC53/78-mCherry and 285 commonly observed contaminants.For in vitro ubiquitination samples, database search was performed against an E. coli database, the sequences of MBP-NAC53/78, GST-HRD1a/b and 285 commonly observed contaminants. Endoprotease trypsin was defined as a protease with a maximum of two missed cleavages. Oxidation of methionine, phosphorylation of serine, threonine, and tyrosine, GlyGly dipeptide on lysine residues, and N‐terminal acetylation were specified as variable modifications. Carbamidomethylation on cysteine was set as a fixed modification. Mass tolerance was set to 4.5 parts per million (ppm) for precursor ions and 20 ppm for fragment ions. Peptide, protein, and modification site identifications were reported at a false discovery rate (FDR) of 0.01, estimated by the target‐decoy approach. The iBAQ (Intensity Based Absolute Quantification) and LFQ (Label‐Free Quantification) algorithms were enabled, as was the “match between runs” option.", "sampleProcessingProtocol" : "For interactome analysis, N. benthamiana leaves were infiltrated with A. tumefaciens for transient expression of corresponding constructs. For bacterial infection, 24h post infiltration leaves were infiltrated with mock solution or bacterial suspension of Pst∆HopQ at OD 0.2. At 30h post A. tumefaciens infiltration, 5g of leaf material was sampled in triplicates for each condition and immuno-pulldown anti-GFP or anti-RFP was performed prior to tdMS analysis. For ubiquitination sites analysis, in vitro trans ubiquitination assay was carried out on MBP-NAC53 and MBP-NAC78 recombinant proteins using GST-HRD1a or GST-HRD1b as E3 ligases catalyzing the ubiquitination reaction prior to tdMS analysis. Proteins were subjected to a NuPAGE 12% gel and in-gel trypsin digestion was done on Coomassie‐stained gel pieces. Peptides mixtures were desalted using C18 Stage tips and run on an Easy‐nLC 1200 system coupled to a Q Exactive HF mass spectrometer. Separation of peptides was done using a 87‐min or 127-min segmented gradient from 10‐33‐50‐90% of HPLC solvent B (80% acetonitrile in 0.1% formic acid) in HPLC solvent A (0.1% formic acid) at a flow rate of 200 nl/min. The seven most intense precursor ions were sequentially fragmented in each scan cycle using higher energy collisional dissociation (HCD) fragmentation. MS and MS/MS spectra were acquired with a resolution of 60k and a maximum injection time of 25 ms and 110 or 220, respectively. The target values were 105 charges for MS/MS fragmentation and 3 × 106 charges for the MS scan. In all measurements, sequenced precursor masses were excluded from further selection for 30 s.", "projectTags" : [ ], "keywords" : [ "Bacterial infection", "Ubiquitination", "Transcription factors", "Ap-ms", "Interactome", "Subcellular localization", "Microtubule depolymerizing factor" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-23", "updatedDate" : "2026-03-12", "submissionDate" : "2026-03-12", "downloadCount" : 6, "avgDownloadsPerFile" : 0.092307694, "botCount" : 3, "hubCount" : 3, "organicCount" : 0, "percentile" : 1, "yearlyDownloads" : [ { "year" : "2026", "count" : 6 } ], "submitters" : [ "Mirita Franz-Wachtel" ], "labPIs" : [ "Suayb Üstün" ], "affiliations" : [ "Ruhr University Bochum Faculty of Biology and Biotechnology Plant Cell Biology" ], "instruments" : [ "Q Exactive HF" ], "softwares" : [ ], "quantificationMethods" : [ "label-free quantification" ], "sampleAttributes" : [ "bacterial infectious disease", "Nicotiana benthamiana", "plant cell", "leaf" ], "organisms" : [ "Nicotiana benthamiana" ], "organismsPart" : [ "Plant cell", "Leaf" ], "diseases" : [ "Bacterial infectious disease" ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition", "Affinity purification coupled with mass spectrometry proteomics", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "20210707_CO_1023ThSt_R05.raw", "20210707_CO_1032SuUe_R11.raw", "20210211_CO_0995SuUe_R14.raw", "20210211_CO_0995SuUe_R05.raw", "20221209_CO_0995SuUe_R29.raw", "20210707_CO_1032SuUe_R02.raw", "20210707_CO_1032SuUe_R04.raw", "20210707_CO_1032SuUe_R10.raw", "checksum.txt", "20210707_CO_1023ThSt_R06.raw", "20210211_CO_0995SuUe_R04.raw", "20210211_CO_0995SuUe_R13.raw", "20210707_CO_1032SuUe_R01.raw", "20210211_CO_0995SuUe_R12.raw", "20210707_CO_1032SuUe_R05.raw", "20210211_CO_0995SuUe_R03.raw", "20210211_CO_0995SuUe_R16.raw", "20210707_CO_1032SuUe_R13.raw", "20221209_CO_0995SuUe_R22.raw", "20210707_CO_1032SuUe_R06.raw", "20210707_CO_1023ThSt_R07.raw", "20210211_CO_0995SuUe_R09.raw", "search_1023_1032.zip", "20210211_CO_0995SuUe_R15.raw", "20210211_CO_0995SuUe_R02.raw", "20210707_CO_1023ThSt_R09.raw", "20210707_CO_1032SuUe_R12.raw", "20221209_CO_0995SuUe_R23.raw", "20210707_CO_1023ThSt_R08.raw", "20210707_CO_1032SuUe_R07.raw", "20221209_CO_0995SuUe_R19.raw", "20210707_CO_1032SuUe_R16.raw", "20221209_CO_0995SuUe_R32.raw", "20210707_CO_1032SuUe_R08.raw", "20210211_CO_0995SuUe_R01.raw", "20221209_CO_0995SuUe_R24.raw", "20210211_CO_0995SuUe_R18.raw", "20210707_CO_1032SuUe_R17.raw", "20221209_CO_0995SuUe_R33.raw", "20210707_CO_1023ThSt_R01.raw", "20221209_CO_0995SuUe_R31.raw", "20221209_CO_0995SuUe_R25.raw", "search_0995_R19R34.zip", "20210707_CO_1032SuUe_R09.raw", "20210211_CO_0995SuUe_R17.raw", "20221209_CO_0995SuUe_R34.raw", "20210707_CO_1032SuUe_R14.raw", "20210707_CO_1032SuUe_R18.raw", "20210211_CO_0995SuUe_R08.raw", "20210707_CO_1023ThSt_R02.raw", "20221209_CO_0995SuUe_R30.raw", "20221209_CO_0995SuUe_R21.raw", "20221209_CO_0995SuUe_R26.raw", "20210211_CO_0995SuUe_R11.raw", "20221209_CO_0995SuUe_R27.raw", "20210211_CO_0995SuUe_R07.raw", "20210707_CO_1023ThSt_R03.raw", "20210707_CO_1032SuUe_R15.raw", "20210211_CO_0995SuUe_R10.raw", "20221209_CO_0995SuUe_R20.raw", "20210211_CO_0995SuUe_R06.raw", "20221209_CO_0995SuUe_R28.raw", "search_0995_R01R18.zip", "20210707_CO_1023ThSt_R04.raw", "20210707_CO_1032SuUe_R03.raw" ], "highlights" : { } }, { "accession" : "PXD075507", "title" : "Unveiling Alternate Pathways for SARS-CoV-2 Infection via Extracellular Vesicle-Mediated Transfer of ACE2 and TMPRSS2", "projectDescription" : "The COVID-19 pandemic, caused by SARS-CoV-2, has underscored the urgency of understanding viral entry mechanisms to develop effective therapeutic strategies. SARS-CoV-2 primarily exploits angiotensin-converting enzyme 2 (ACE2) as its entry receptor and relies on the serine protease TMPRSS2 to prime its spike protein, enabling membrane fusion and infection. Traditionally, TMPRSS2 has been described as a cell surface protein, but our study reveals that in human lung epithelial cells, TMPRSS2 is largely absent from the plasma membrane and instead resides intracellularly. We show that TMPRSS2 is secreted together with ACE2 in extracellular vesicles (EVs) from lung epithelial cells, which are subsequently taken up by non-epithelial cells, specifically alveolar macrophages, endothelial cells, and pericytes, that do not express TMPRSS2 or ACE2 mRNA under homeostatic conditions. This EV uptake deposits ACE2 and TMPRSS2 protein onto recipient cells, equipping them for SARS-CoV-2 entry. By transferring these viral entry proteins, EVs expand the spectrum of susceptible cell types in the lung, offering a new explanation for how the virus can infect diverse cell populations and cause widespread tissue damage. Identifying EVs as vehicles for delivering functional ACE2 and TMPRSS2 across cell types reveals previously unrecognized pathway of viral entry with important implications for COVID-19 pathogenesis. These findings open new avenues for therapeutic intervention aimed at disrupting EV-mediated protein transfer, potentially limiting viral dissemination and severity, and may also represent a generalizable mechanism exploited by other viral pathogens, highlighting the potential relevance of EV-mediated protein transfer beyond SARS-CoV-2.", "dataProcessingProtocol" : "The DIA-NN algorithm was used identify peptides/proteins and to extract intensity information from DIA data [ref 31768060]. Data were searched against the Homo sapiens reference proteome sequences in the UniProt database (one protein sequence per gene, downloaded on August 23, 2023). Search parameters included a fixed modification for carbamidomethyl cysteine and variable modifications for N-terminal protein acetylation and methionine oxidation. All other search parameters were DIA-NN factory defaults. Statistical analysis of proteomics data was conducted utilizing the MSstats package in R [ref 24794931]. All data were normalized by equalizing median intensities, the summary method was Tukey’s median polish, and the maximum quantile for deciding censored missing values was 0.999. For protein abundance analyses, only the top 20 peptide features per protein were considered.", "sampleProcessingProtocol" : "Samples were incubated in a buffer containing 2 M urea, 50 mM Tris-HCl, pH 8.0, and 1 mM DTT at 37 °C for 30 min. Iodoacetamide was added to a final concentration of 3 mM and samples were incubated at room temperature for 45 min in the dark. DTT was added to a final concentration of 3 mM and 750 ng of trypsin (trypsin gold, Promega) was added to each sample, followed by overnight incubation at 37 °C with shaking. The supernatant was transferred to a fresh tube and an additional 500 ng trypsin added to the supernatant, followed by incubation for 2 h at 37 °C. Digested samples were desalted on BioPureSPN Mini C18 SPE columns (Nest Group) according to the manufacturer’s protocol. Samples were dried by vacuum centrifugation and resuspended in 0.1% formic acid (FA) for mass spectrometry analysis.", "projectTags" : [ ], "keywords" : [ "Sars-cov-2", "Ace2", "Tmprss2" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-11", "updatedDate" : "2026-03-11", "submissionDate" : "2026-03-11", "downloadCount" : 79, "avgDownloadsPerFile" : 6.076923, "botCount" : 15, "hubCount" : 64, "organicCount" : 0, "percentile" : 4, "yearlyDownloads" : [ { "year" : "2026", "count" : 79 } ], "submitters" : [ "Jeffrey Johnson" ], "labPIs" : [ "Jeffrey Johnson" ], "affiliations" : [ "Icahn School of Medicine at Mount Sinai" ], "instruments" : [ "Orbitrap Eclipse" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "EC20250725_23.raw", "EC20250725_17.raw", "20250730-YC2-ab-keys-forPRIDE.xlsx", "EC20250725_19.raw", "EC20250725_20.raw", "EC20250725_24.raw", "EC20250725_26.raw", "checksum.txt", "20250728YC02-IPabDIAnn.tsv", "EC20250725_22.raw", "EC20250725_18.raw", "EC20250725_21.raw", "EC20250725_25.raw" ], "highlights" : { } }, { "accession" : "PXD075505", "title" : "Unveiling Alternate Pathways for SARS-CoV-2 Infection via Extracellular Vesicle-Mediated Transfer of ACE2 and TMPRSS2", "projectDescription" : "The COVID-19 pandemic, caused by SARS-CoV-2, has underscored the urgency of understanding viral entry mechanisms to develop effective therapeutic strategies. SARS-CoV-2 primarily exploits angiotensin-converting enzyme 2 (ACE2) as its entry receptor and relies on the serine protease TMPRSS2 to prime its spike protein, enabling membrane fusion and infection. Traditionally, TMPRSS2 has been described as a cell surface protein, but our study reveals that in human lung epithelial cells, TMPRSS2 is largely absent from the plasma membrane and instead resides intracellularly. We show that TMPRSS2 is secreted together with ACE2 in extracellular vesicles (EVs) from lung epithelial cells, which are subsequently taken up by non-epithelial cells, specifically alveolar macrophages, endothelial cells, and pericytes, that do not express TMPRSS2 or ACE2 mRNA under homeostatic conditions. This EV uptake deposits ACE2 and TMPRSS2 protein onto recipient cells, equipping them for SARS-CoV-2 entry. By transferring these viral entry proteins, EVs expand the spectrum of susceptible cell types in the lung, offering a new explanation for how the virus can infect diverse cell populations and cause widespread tissue damage. Identifying EVs as vehicles for delivering functional ACE2 and TMPRSS2 across cell types reveals previously unrecognized pathway of viral entry with important implications for COVID-19 pathogenesis. These findings open new avenues for therapeutic intervention aimed at disrupting EV-mediated protein transfer, potentially limiting viral dissemination and severity, and may also represent a generalizable mechanism exploited by other viral pathogens, highlighting the potential relevance of EV-mediated protein transfer beyond SARS-CoV-2.", "dataProcessingProtocol" : "The DIA-NN algorithm was used identify peptides/proteins and to extract intensity information from DIA data [ref 31768060]. Data were searched against the Homo sapiens reference proteome sequences in the UniProt database (one protein sequence per gene, downloaded on August 23, 2023). Search parameters included a fixed modification for carbamidomethyl cysteine and variable modifications for N-terminal protein acetylation and methionine oxidation. All other search parameters were DIA-NN factory defaults. Statistical analysis of proteomics data was conducted utilizing the MSstats package in R [ref 24794931]. All data were normalized by equalizing median intensities, the summary method was Tukey’s median polish, and the maximum quantile for deciding censored missing values was 0.999. For protein abundance analyses, only the top 20 peptide features per protein were considered.", "sampleProcessingProtocol" : "Samples were incubated in a buffer containing 2 M urea, 50 mM Tris-HCl, pH 8.0, and 1 mM DTT at 37 °C for 30 min. Iodoacetamide was added to a final concentration of 3 mM and samples were incubated at room temperature for 45 min in the dark. DTT was added to a final concentration of 3 mM and 750 ng of trypsin (trypsin gold, Promega) was added to each sample, followed by overnight incubation at 37 °C with shaking. The supernatant was transferred to a fresh tube and an additional 500 ng trypsin added to the supernatant, followed by incubation for 2 h at 37 °C. Digested samples were desalted on BioPureSPN Mini C18 SPE columns (Nest Group) according to the manufacturer’s protocol. Samples were dried by vacuum centrifugation and resuspended in 0.1% formic acid (FA) for mass spectrometry analysis.", "projectTags" : [ ], "keywords" : [ "Sars-cov-2", "Ace2", "Tmprss2" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-25", "updatedDate" : "2026-03-11", "submissionDate" : "2026-03-11", "downloadCount" : 1, "avgDownloadsPerFile" : 0.11111111, "botCount" : 0, "hubCount" : 1, "organicCount" : 0, "percentile" : 0, "yearlyDownloads" : [ { "year" : "2026", "count" : 1 } ], "submitters" : [ "Jeffrey Johnson" ], "labPIs" : [ "Jeffrey Johnson" ], "affiliations" : [ "Icahn School of Medicine at Mount Sinai" ], "instruments" : [ "Orbitrap Eclipse" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "report20241018TC1_abDIAnn.tsv", "EC20241023-04.raw", "EC20241023-09.raw", "checksum.txt", "EC20241023-05.raw", "20241029-YWC1-ab-keys-forPRIDE.xlsx", "EC20241023-08.raw", "EC20241023-07.raw", "EC20241023-06.raw" ], "highlights" : { } }, { "accession" : "PXD075513", "title" : "Adipose Dicer-1 modulates systemic insulin signaling and longevity via a miR-8–Aop–Dilp6 axis", "projectDescription" : "This project examines how adipose tissue regulates systemic metabolism and aging in Drosophila melanogaster through the miRNA-processing enzyme Dicer-1 (Dcr-1). Proteomic profiling of fat bodies from Dcr-1 heterozygous flies was performed to identify molecular changes associated with reduced Dcr-1 activity. The dataset reveals metabolic and stress-response remodeling consistent with reduced insulin/IGF signaling, supporting a model in which decreased Dcr-1 increases Dilp6 expression in the fat body and suppresses Dilp2 secretion from brain insulin-producing cells, promoting longevity.", "dataProcessingProtocol" : "MS data were analyzed using Proteome Discoverer v2.4.1.15 with standard workflows. Spectrawere searched against the Drosophila melanogaster UNIPROT database (UP000000803) with precursor and fragment mass tolerances of 10 ppm and 0.02 Da, respectively, allowing up to twomissed cleavages.", "sampleProcessingProtocol" : "We compared fat body samples from four biological replicates of wild-type (w1118) and dcr-1MI13376 heterozygous larvae. Fat bodies were dissected and processed for proteomic analysis. Peptides were separated on a nanoHPLC Ultimate 3000 system (Thermo Scientific) using an EASY-Spray ES903 column (50 cm × 50 μm ID, PepMap RSLC C18) at a flow rate of 300 nL/min. Solvent A consisted of 0.1% formic acid in water, and solvent B of 0.1% formic acid in acetonitrile. The gradient was as follows: 4–30% B for 114 min, 30–80% B for 14 min, and 80% B for 2 min. Mass spectrometry was performed on a Q-Exactive HF (Thermo Scientific) with the following settings: full scan range m/z 375–2000, resolution 120,000 (at m/z 200), AGC target 1 × 10^6, and maximum injection time 100 ms. The 15 most intense ions were selected for MS/MS fragmentation using a normalized collision energy of 28 eV. MS/MS scans were acquired at 30,000 resolution with an AGC target of 5 × 10^5, intensity threshold 1.5 × 10^5, isolation window 1.4 m/z, and maximum injection time 55 ms. Singly charged, unassigned, and ≥6+ ions were excluded, and dynamic exclusion was set to 30 s.", "projectTags" : [ ], "keywords" : [ "Oxidative stress", "Adipose tissue", "Inter organ communication", "Mirna", "Insulin like peptides" ], "doi" : "10.6019/PXD075513", "submissionType" : "COMPLETE", "publicationDate" : "2026-04-13", "updatedDate" : "2026-03-11", "submissionDate" : "2026-03-11", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Andrés Dekanty" ], "labPIs" : [ "Andres Dekanty" ], "affiliations" : [ "Laboratorio de Biología Molecular - Instituto de Agrobiotecnología del Litoral - CONICET - Santa Fe,Argentina" ], "instruments" : [ "Q Exactive HF" ], "softwares" : [ ], "quantificationMethods" : [ "label-free quantification" ], "sampleAttributes" : [ "Drosophila melanogaster (Fruit fly)", "fat body" ], "organisms" : [ "Drosophila melanogaster (fruit fly)" ], "organismsPart" : [ "Fat body" ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.1073/PNAS.2525327123" ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "D1.raw", "D4.raw", "D3.raw", "D2.raw", "W2.raw", "LFQ_Turno262376.mzTab", "W1.raw", "W3.raw", "W4.raw" ], "highlights" : { } }, { "accession" : "PXD075514", "title" : "Glycan-based Biological Degraders Targeting the Cytokine Immune Axis", "projectDescription" : "The project investigates targeted degradation of IL-6 and sIL6-R using bispecific Biological Degraders (BioDegs) that enable receptor-mediated uptake via the asialoglycoprotein receptor (ASGPR). Various IL-6(R)–binding scaffolds were modified with triantennary N-acetylgalactosamine (TGN) to promote lysosomal targeting. Mass spectrometry was used to determine the glycan-to-protein ratio after modification of the different binders, providing analytical characterization of the conjugates and confirming successful generation of BioDeg constructs.", "dataProcessingProtocol" : "Mass spectra were acquired in positive ion mode over an m/z range of 500–4000 using full-scan acquisition. The spectra rate was set to 2 Hz, and rolling average smoothing was applied with a 2× setting. Mass spectra were deconvoluted using the MaxEnt algorithm implemented in Compass DataAnalysis 4.2 software (Bruker Daltonics).", "sampleProcessingProtocol" : "Binder molecules including siltuximab, tocilizumab, sIL-6R, and a VHH against IL-6 were modified via TGN conjugation to enable lysosomal targeting. Purified proteins (>1 mg/mL) were reacted with Tris-GalNAc-PEG5-sulfo-NHS Ester (Sussex Research, MV1000054, 5 mg/mL in DMSO) in 1× PBS. Molar ratios of 1:1, 1:5, 1:10, and 1:25 were selected based on the number of available lysine residues. Reactions were incubated overnight at room temperature under rotation and subsequently quenched with Tris/HCl (pH 9.0) to a final concentration of 10 mM. Excess reagent was removed by three rounds of centrifugal filtration using Amicon® Ultra units (Merck Millipore) with MWCOs of 30 kDa for antibody-based BioDegs and 10 kDa for sIL-6R and VHH constructs. Samples were washed three times with 300 µL of 1× PBS to remove unbound components and exchange the buffer. The intact masses of proteins were determined by electrospray ionization mass spectrometry (ESI-MS) using a quadrupole time-of-flight (QTOF) mass spectrometer (Maxis, Bruker Daltonics) following desalting via high-performance liquid chromatography (HPLC, Agilent Technologies). A total of 200 µL of protein sample at a concentration of 0.25 µM was loaded onto a reversed-phase trapping column (0.8 × 2 mm, Poros R1; Applied Biosystems). After washing for 3 minutes with 0.3% formic acid at a flow rate of 300 µL/min, proteins were eluted using a mobile phase consisting of 40% isopropanol, 5% acetonitrile, and 0.3% formic acid at a flow rate of 40 µL/min.", "projectTags" : [ ], "keywords" : [ "Biological degraders", "Lysosomal degradation", "Tri-n-acetylgalactosamine", "Targeted protein degradation", "Sil-6r", "Asgpr", "Il-6" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-17", "updatedDate" : "2026-03-11", "submissionDate" : "2026-03-11", "downloadCount" : 26, "avgDownloadsPerFile" : 0.8965517, "botCount" : 7, "hubCount" : 18, "organicCount" : 1, "percentile" : 2, "yearlyDownloads" : [ { "year" : "2026", "count" : 26 } ], "submitters" : [ "Marcin Luzarowski" ], "labPIs" : [ "Marcin Luzarowski" ], "affiliations" : [ "Core Facility for Mass Spectrometry and Proteomics, Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany" ], "instruments" : [ "maXis 4G" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Top-down proteomics" ], "sdrf" : "", "projectFileNames" : [ "sample_4_1_01_3771.d.rar", "sample_3_1_01_3769.d.rar", "sample_19_1_01_3756.d.rar", "sample_1_1_01_3766.d.rar", "sample_7.xy", "sample_5.xy", "sample_9.xy", "sample_1.xy", "sample_17.xy", "sample_3.xy", "sample_19.xy", "sample_18_1_01_3753.d.rar", "sample_8_1_01_3761.d.rar", "sample_10_1_01_3763.d.rar", "sample_8.xy", "sample_6.xy", "sample_7_1_01_3760.d.rar", "sample_9_1_01_3762.d.rar", "sample_2.xy", "sample_5_1_01_3772.d.rar", "sample_16.xy", "sample_4.xy", "sample_2_1_01_3767.d.rar", "sample_16_1_01_3751.d.rar", "sample_17_1_01_3755.d.rar", "sample_6_1_01_3759.d.rar", "MS_Samples.xlsx", "sample_18.xy", "sample_10.xy" ], "highlights" : { } }, { "accession" : "PXD075509", "title" : "E.coli total RNA digests higer-collision dissociation energies test in LC-MS/MS", "projectDescription" : "Four open-source software suites for the analysis of oligonucleotide data from tandem MS have been reported. These tools are all based on matching theoretical spectral libraries generated by silico digestion. While some software publications have included simple comparisons with previously published tools, a discussion of oligonucleotide identification within complex backgrounds is lacking. In this study, we acquired multiple complex oligonucleotide datasets using an Orbitrap instrument to evaluate various search engines (Ariadne, RNAModMapper, NASE and Pytheas) and assess their performance in terms of accuracy, false discovery rate (FDR), performance at certain FDR, consensus of oligonucleotide identification, consistency of outputs in chemical measurements, and unknown modification discovery.In this study, we prepared two sets of samples, which involve three RNase T1 digested and three RNase A digested E. coli total RNA. Then, digests were analyzed by LC-MS/MS.", "dataProcessingProtocol" : "Ariadne, RNAModMapper (RAMM), NucleicAcidSearchEngine (NASE) and Pytheas were involved in the evaluation. The raw LC-MS/MS files were converted to either mgf or mzML format following software’s requirement. For software parameters’ settings, mass error was uniformed, which was set as 10 ppm (Δ 0.01 m/z) for precursor ions m/z and 15 ppm (Δ 0.05 m/z) for fragment ions m/z. In silico enzyme digestion miss cleavage was set to 2 for all applied samples. All rest of parameters were maintained as defaults.", "sampleProcessingProtocol" : "E. coli DH5α strain grown at 37 °C in the LB medium. Cells were precipitated by centrifugation and washed with PBS. 1 mL TRIzol was added to the E. coli and freeze under -80°C .TRIzol extraction method was applied to total RNA extraction. In brief, E. coli cells were lysed by adding TRIzol reagent at a ratio of 1:10 (w/v: mg/µL), followed by thorough mixing. The mixture was incubated on ice for 5 minutes, after which chloroform (one-fifth of the volume of TRIzol reagent used) was added and mixed vigorously. The sample was then centrifuged at 12,000 × g and 4 °C for 10 minutes. The upper aqueous phase was carefully transferred to a new tube, and isopropanol (equivalent to 0.7 volumes of the aqueous phase) was added. After thorough mixing, the sample was allowed to stand for 10 minutes and then centrifuged at 12,000 × g and 4 °C for 10 minutes to pellet the RNA. The RNA pellet was washed once with 75% ethanol, the supernatant was removed, and the pellet was air-dried briefly. Finally, the RNA was dissolved in RNase-free water. RNAs were heat-denatured for 5 min and digested by RNase T1 or A for 1 hour at 37 °C in RNase T1 buffer (100 mM Tris-HCl, pH 7.5 and 1mM EDTA) or RNase A buffer (30 mM ammonium acetate, pH 7.0). Digests were then heat-denatured for enzyme inactivation and 20 μg of each sample was used for single injection. Although identical RNA samples were used, different enzymatic digestion generated completely distinct oligonucleotide mixtures. A Vanquish UHPLC system coupled with an Orbitrap Lumos mass spectrometer (ThermoFisher Scientific) was used for oligos analysis. Separation was achieved on an ACQUITY UPLC Oligonucleotide BEH C18 column (130 Å, 1.7 μm, 2.1 mm × 150 mm, Waters Corporation). The column temperature was maintained at 65 °C, and the injection volume was 10 μL. Each sample was analyzed in technical triplicate. The mobile phase A was 1% Hexafluoroisopropanol (HFIP) and 0.1% N,N-Diisopropylethylamine (DIPEA) in water, and mobile phase B was 50% methanol with water. The LC flow rate was set to 0.2 mL/min. The gradient shows as follows: 0–3 min, isocratic at 3% B; 3–5 min, linearly increased from 3% to 18% B; 5–15 min, linearly increased from 18% to 28% B; 15–28 min, linearly increased from 28% to 40% B; 28–40 min, linearly increased from 40% to 60% B; 40–40.1 min, linearly increased from 60% to 80% B; 40.1–42.5 min, isocratic at 80% B; 42.5–42.6 min, linearly return to 3% B; 42.6–52.6 min, isocratic at 3% B. The mass spectrometer was operated in negative ion mode with the heated electrospray ionization source (HESI). Data acquisition was performed in Full MS/dd-MS² mode. Full MS scans (750–2000 m/z) were acquired in the Orbitrap at 30,000 resolutions (AGC 125%, 50 ms MIT, 2 microscans). DDA parameters included: top 10 precursor ions, charge states 2–30, 1 × 10⁵ intensity threshold, and 10-second dynamic exclusion. MS/MS spectra were acquired at 30,000 resolutions. For gradient analysis, HCD was varied from 15% to 31% in 4% increments). Each energy has three technical repeats and contains two types of digests by RNase T1 and A.", "projectTags" : [ ], "keywords" : [ "E.coli rna", "Hcd", "Lc-ms/ms", "Oligonucleotide" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-11", "updatedDate" : "2026-03-11", "submissionDate" : "2026-03-11", "downloadCount" : 53, "avgDownloadsPerFile" : 1.4722222, "botCount" : 43, "hubCount" : 5, "organicCount" : 5, "percentile" : 3, "yearlyDownloads" : [ { "year" : "2026", "count" : 53 } ], "submitters" : [ "Jing Feng" ], "labPIs" : [ "Xiang Zhang" ], "affiliations" : [ "University of Louisville, Department of Chemistry, Louisville, KY 40208, USA" ], "instruments" : [ "Orbitrap Fusion Lumos" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "cell culture", "Escherichia coli" ], "organisms" : [ "Escherichia coli" ], "organismsPart" : [ "Cell culture" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "Pytheas.csv", "NASE.csv", "Ecoli_rRNA.fasta", "15HCD_3.mzML", "19HCD_2.mzML", "31HCD_1.mzML", "27HCD_2.mzML", "checksum.txt", "Ariadne.csv", "19HCD_3.mzML", "15HCD_2_A.mzML", "15HCD_1_A.mzML", "15HCD_1.mzML", "27HCD_2_A.mzML", "15HCD_3_A.mzML", "27HCD_1_A.mzML", "27HCD_3_A.mzML", "31HCD_3.mzML", "19HCD_3_A.mzML", "23HCD_2_A.mzML", "RAMM.csv", "23HCD_3_A.mzML", "27HCD_3.mzML", "23HCD_1_A.mzML", "23HCD_1.mzML", "23HCD_3.mzML", "19HCD_1_A.mzML", "19HCD_2_A.mzML", "19HCD_1.mzML", "31HCD_2.mzML", "31HCD_3_A.mzML", "31HCD_2_A.mzML", "31HCD_1_A.mzML", "15HCD_2.mzML", "27HCD_1.mzML", "23HCD_2.mzML" ], "highlights" : { } }, { "accession" : "PXD075504", "title" : "Analysis of lymphoma EV and serum samples", "projectDescription" : "Detecting extracellular vesicle-related markers in EV samples extracted from human lymphoma cell lines and EVs enriched from serum samples.", "dataProcessingProtocol" : "Data analysis consisted of protein identifications and label free quantifications of protein abundances. Data was analyzed by Spectronaut software (Biognosys).", "sampleProcessingProtocol" : "Isolated EVs were lysed in a buffer containing 8 M urea, 0.5% NP40, 150 mM NaCl, and 1X protease inhibitors for 30 minutes on ice. The samples were sonicated for 5 minutes (30 s on and 30 s off), and the proteins were precipitated using 4-5 volumes of cold acetone overnight at -20 °C. The samples were cleared by centrifugation for 15 minutes at 16,000 × g and 4 °C, and the pellet was dissolved in 100 µL of 8 M urea in 50 mM ammonium bicarbonate. All of the samples were used for in-solution digestion. The samples were reduced with 10 mM DTT for 1 hour at 37 °C and alkylated with 40 mM IAA for 1 hour in the dark. The alkylation was quenched with 40 mM DTT, and the urea concentration was diluted by adding 900 µL of 50 mM ammonium bicarbonate. Sequencing-grade trypsin was added to each sample in a 1:30 ratio, and the samples were incubated overnight at 37 °C. After digestion, the peptide samples were acidified to 1% trifluoroacetic acid (TFA) concentration (pH 2), desalted using a Sep-Pak tC18 96-well plate (Waters), and evaporated to dryness. For MagNet enrichment, equal volumes of the samples were mixed with binding buffer to a final volume of 50 µL. Then, 2 µL of MagReSyn SAX beads (ReSyn Biosciences) were added and mixed by pipetting. Reduction, alkylation, and wash steps were performed using an Opentrons pipetting robot (Opentrons, New York, United States) following the Proteomics Facility’s MagNet enrichment protocol. In the final step, sequencing-grade modified trypsin (Promega) was added, and the sample plate was incubated overnight at +37 °C. On the following day, half of each sample was loaded onto Evotip Pure tips (Evosep) and analyzed using a TimsTOF mass spectrometer. The LC-ESI-MS/MS analysis was performed on Evosep One HPLC system (Evosep, Odense, Denmark) coupled to the timsTOF fleX mass spectrometer (Bruker, Bremen, Germany) equipped with a CaptiveSpray nano-electrospray ionization source. Peptides were separated inline on a 15 cm Evosep Performance column (150 μm x 15 cm, 1.5 μm C18-beads, EV1137). The mobile phase consisted of water with 0.1% formic acid (solvent A) and 0.1% formic acid/99.9% acetonitrile (v/v) (solvent B). Samples were analyzed with 44 min 30 samples per day method. Samples were analyzed by a data independent acquisition (DIA) LC-MS/MS method. MS data was acquired automatically by using Compass 2025 software (Bruker). The DIA method covered a m/z range from 350 to 1100 and included two ion mobility (IM) windows per dia-PASEF scan with variable isolation window widths adjusted to the precursor densities. Twenty-five dia-PASEF scans were deployed, and IM range was set to 1.3 and 0.6 Vcm-2. Accumulation and ramp times were specified as 100 ms.", "projectTags" : [ ], "keywords" : [ "Human lymphoma extracellular vesicle" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-26", "updatedDate" : "2026-03-11", "submissionDate" : "2026-03-11", "downloadCount" : 4, "avgDownloadsPerFile" : 0.36363637, "botCount" : 0, "hubCount" : 4, "organicCount" : 0, "percentile" : 0, "yearlyDownloads" : [ { "year" : "2026", "count" : 4 } ], "submitters" : [ "Saara Hämälistö" ], "labPIs" : [ "Saara Hämälistö" ], "affiliations" : [ "University of Turku, Institute of Biomedicine, Finland" ], "instruments" : [ "Orbitrap Exploris 480", "timsTOF fleX" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "cell culture", "Homo sapiens (Human)", "blood serum" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Cell culture", "Blood serum" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-independent acquisition" ], "sdrf" : "", "projectFileNames" : [ "250306_SUDHL_4.raw", "checksum.txt", "Serum_EVs_SEC.xlsx", "250306_U_2932.raw", "250306_OCI_LY7.raw", "Cell_line_EVs_SEC.xlsx", "SEC_EV_folders_zipped.zip", "Plain_serum_digestion.xlsx", "250306_Riva.raw", "MAgNet_and_plain_serum_files_zipped.zip", "Serum_MAgNet_enrichment.xlsx" ], "highlights" : { } }, { "accession" : "PXD075511", "title" : "Proteomic data of Listeria monocytogenes", "projectDescription" : "Listeria monocytogenes is an important food-borne pathogen that is responsible for contamination of a variety of food products. It causes listeriosis which is one of the most severe and serious diseases whose symptoms might include nausea and diarrhea. Also because of its ability to adhere to industrials’ surfaces, it is difficult for them to come up against this danger. Although there are researches in L. monocytogenes proteome, most of them are based on the study of a single strain, yet little is known about the proteome of more of the microbe’s strains. By studying the proteome of the microorganism with state-of-the-art mass spectrometry technology in terms of proteomics, we will be able to have an overall picture of the pathogenicity of the bacterium, as this is largely based on its ability to adapt to certain environments and to project its toxicity in them. To address that we provide a dataset of 2227 different proteins from 4 strains of L. monocytogenes, grouped by their molecular function and their biological process. The identified proteins confirm the complex molecular mechanisms of the bacterium.", "dataProcessingProtocol" : "The timsTOF raw data was analysed in Proteoscape environment using the Spectronaut software version 19, searching against the reference Listeria monocytogenes database retrieved from Uniprot in the library free mode of the software, allowing up to two tryptic missed cleavages. Default settings have been used with oxidation of methionine residues and acetylation of the protein N-termini set as variable modifications and carbamidomethylation of cysteine residues as fixed modification. N-terminal methionine excision was also enabled. The match between runs (MBR) feature was used for all analyses and the output (precursor and protein group) was filtered at 1% FDR.", "sampleProcessingProtocol" : "30 µl of the protein extract was taken for further analysis. The mixture was subjected to alternating heating and sonication twice. Subsequently, the sample was centrifuged at 17,000 × g for 15 min and the supernatant was treated according to Hughes’ single-pot solid-phase enhanced sample preparation (Sp3) method [6] without acidification and protein alkylation was performed by adding cysteine to 100 mM iodoacetamide. Digestion was performed at 1200 rpm and 37 °C using 0.5 µg trypsin-platinum mixture in 100 mM TEAB buffer. The next day, magnetic particles were removed, and the peptide sample was further purified using the Sp3 peptide purification kit as previously described [6]. Briefly, 30 μl of the peptide mixture was added to 1.2 ml of acetonitrile (ACN) and they were mixed by inversion for 30 minutes, then the tubes were placed on a magnetic rack. The supernatant was removed and the remaining sample was washed twice with 200 µl of 100% ACN, removing the bound peptide magnetic particles with each wash. Subsequently, 50 µl of water was added to elute the peptide and magnetic particles. The elution buffer was concentrated using SpeedVac. Finally, the dried peptide was dissolved in buffer A, sonicated for 5 minutes, and the absorbance was measured at 280 nm using a Nanodrop spectrophotometer to determine the peptide concentration. 1-D nanoLC-MS/MS analysis The peptide content of the samples was then analyzed using a NanoElute 2 liquid chromatography system coupled to a timsTOF Ultra2 mass spectrometer (Bruker Daltonik GmbH). Briefly, peptides were separated on a temperature-controlled (50 °C) Pepsep column (25 cm × 75 µm, 1.9 µm particle size, Bruker) and ionized using a Captive Spray Ultra-2 ion source. Peptide elution from the column was performed using a 30-minute gradient elution. The mass spectrometer was in dia-PASEF mode, using standard methods for small sample volumes (mobilities 0.64–1.45, cycle time 0.96 s). Complete MS data were acquired in the range of 400–1000 m/z. The ion mobility settings included three isolation windows, all of which were covered in a single TIMS scan. Eight TIMS scans were required to cover all 24 windows.", "projectTags" : [ ], "keywords" : [ "Proteomics protein content nanolc-ms/ms bottom-up proteomics bacteria listeria monocytogenes timstof ultra2" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-07", "updatedDate" : "2026-03-11", "submissionDate" : "2026-03-11", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Panagiotis Skandamis" ], "labPIs" : [ "Dr. Panagiotis Skandamis" ], "affiliations" : [ "Laboratory of Food Quality and Hygiene, Department of Food Science and Human Nutrition, Agricultural University of Athens, 118 55 Athens Greece" ], "instruments" : [ "timsTOF Ultra 2" ], "softwares" : [ ], "quantificationMethods" : [ "Relative quantification" ], "sampleAttributes" : [ "Listeria monocytogenes EGD-e" ], "organisms" : [ "Listeria monocytogenes egd-e" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-independent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "Key.xlsx", "checksum.txt", "Listeria_monocytogenes_Results.tsv", "Listeriamonocytogenes.7z" ], "highlights" : { } }, { "accession" : "PXD075517", "title" : "A proximity labelling derived luminal proteome of the Trypanosoma brucei endoplasmic reticulum, Golgi apparatus and nuclear membrane", "projectDescription" : "We employed proximity labelling to derive a proteome of the luminal content of the Trypanosoma brucei endoplasmic reticulum (ER) and Golgi apparatus. We exploited the abundant ER chaperone BiP (Binding-immunoglobulin protein) as ER BioID bait and compare to a truncated version of BiP (termed BiPN) devoid of ER retention and thus trafficked to the Golgi apparatus, as well as parental controls. Our approach covers the two major life cycle stages of T. brucei, i.e., the procyclic form (PCF) and the bloodstream form (BSF). Additionally, we probed the inner nuclear membrane which can be viewed as a specialised ER domain separated by the nuclear pore, via the membrane integral nucleoporin NUP65 as BioID bait in PCF. Altogether, our combined approaches devise an inventory of the T. brucei secretory pathway that complements existing subcellular protein maps derived by proteomics and imaging.", "dataProcessingProtocol" : "Spectra were processed using the intensity-based label-free quantification (LFQ) in MaxQuant version 2.1.3.0 (PMID: 19029910; PMID: 24942700) searching the T. brucei brucei 927 annotated protein database (release 64) from TriTrypDB (PMID:19843604). False discovery rates (FDR) of 0.01 were calculated at the levels of peptides, proteins and modification sites based on the number of hits against the reversed sequence database. Sample layout: (1) BioID procyclic-form T. brucei with C-terminal TurboID-HA-MDDL fusions for BiP (triplicate): (1.1) BIP_PCF_1.raw, (1.2) BIP_PCF_2.raw, (1.3) BIP_PCF_3.raw (2) BioID bloodstream-form T. brucei with C-terminal TurboID-HA-MDDL fusions for BiP (triplicate): (2.1) BIP_BSF_1.raw, (2.2) BIP_BSF_2.raw, (2.3) BIP_BSF_3.raw (3) BioID procyclic-form T. brucei BiPN-TurboID-HA expression (triplicate): (3.1) BIPN_PCF_1.raw, (3.2) BIPN_PCF_2.raw, (3.3) BIPN_PCF_3.raw (4) BioID bloodstream-form T. brucei BiPN-TurboID-HA expression (triplicate): (4.1) BIPN_BSF_1.raw, (4.2) BIP_BSF_2.raw, (4.3) BIP_BSF_3.raw (5) parental controls boodstream-form T. brucei (triplicate): (5.1) BioID_BSF_wtcontrol_1.raw, (5.2) BioID_BSF_ wtcontrol _2.raw, (5.3) BioID_BSF_ wtcontrol _3.raw) (6) parental controls procyclic-form T. brucei (triplicate): deposited in PXD031245 - (6.1) BioID_PCF_wtcontrol_1.raw, (6.2) BioID_PCF_ wtcontrol _2.raw, (6.3) BioID_PCF_ wtcontrol _3.raw) (7) BioID procyclic-form T. brucei with C-terminal TurboID-HA fusions for NUP65 (duplicate) (7.1) NUP65C_1.raw (7.2) NUP65C_2.raw", "sampleProcessingProtocol" : "BiP and NUP65 BioID fusion proteins were expressed from the endogenous locus with a TurboID-HA tandem tag fused to the C-terminus of each respective target protein, by modifying one allele via a PCR-based approach reliant on a (modified) version of the pPOT vector system (PMID: 25567099). For the BiP gene (Tb927.11.7510), TurboID-HA was inserted upstream the coding sequence of the BiP C-terminal ER-retention signal-tetrapeptide (MDDL). BiPN was expressed using the pDEX system fusing 2HA-TurboID to BiP residue 415 (PMID: 17512617). A total 5x10^8 cells were used in each replicate. Cells were harvested at 1,400 g, washed once with serum-free medium and pellets rapidly frozen in liquid nitrogen and stored at -80°C. For isolation of biotinylated proteins, each cell pellet was resuspended in 1 ml lysis buffer (0.5% octylphenoxypolyethoxyethanol (IGEPAL), 0.1 M piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES)-NaOH pH 6.9, 2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1 mM MgSO4, 0.1 mM ethylenediaminetetraacetic acid (EDTA), cOmplete EDTA-free protease inhibitor cocktail (Roche)) and incubated for 15 min at room temperature in an orbital mixer. The soluble and non-soluble fractions were separated by centrifugation (14,000 g, 5 min, 4°C) and the soluble fraction incubated with 100 μl streptavidin-linked Dynabeads (MyOne Streptavidin C1, Thermofisher) for 1 hour at 4°C under gentle mixing. Beads were washed twice in 1 ml buffer 1 (2% (w/v) SDS in water) once in 1 ml buffer 2 (0.1% (w/v) deoxycholate, 1% Triton X-100, 1 mM EDTA, 50 mM HEPES pH7.5, 500 mM NaCl), once in 1ml buffer 3 (250 mM LiCl, 0.5% IGEPAL, 0.5% (w/v) deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) and once in 1 ml buffer 4 (50 mM Tris-HCl pH 7.4, 50 mM NaCl); each washing step was 8 minutes at room temperature (RT) under orbital shaking. Beads were then prepared for tryptic digestion by washing three times in 500 µl ice-cold 50 mM NH4HCO3, resuspension in 40 µl of the same buffer supplemented with 10 mM dithiothreitol and incubation in a thermomixer at RT for 1h. Iodoacetamide was added to a concentration of 20 mM, followed by incubation in the dark at RT for 30 min. Finally, 5 μg/ml proteomics-grade trypsin (SOLu-Trypsin, Sigma) and 1 mM biotin was added to the beads. The digest was done overnight at 30°C in a thermomixer (1000 rpm). After removal of the first eluate, beads were resuspended in 50 μl 50 mM NH4HCO3 supplemented with 10 mM dithiothreitol and 5 μg/ml trypsin and incubated in a thermomixer at 37°C for 1h. The eulate was combined with the first eluate and both were dried in a speed-vac. LoBind tubes (Eppendorf) were used throughout. BioID eluted peptides were resuspended in 50 mM NH4HCO3 and passed over C18 stage tip columns as described (PMID: 17703201), then analysed by liquid chromatography-tandem mass spectrometry (LC-MSMS) on an Ultimate3000 nano rapid separation LC system (Dionex) coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific).", "projectTags" : [ ], "keywords" : [ "Trypanosoma brucei", "Nuclear envelope", "Secretory pathway", "Endoplasmic reticulum", "Golgi apparatus" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-05", "updatedDate" : "2026-03-11", "submissionDate" : "2026-03-11", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Martin Zoltner" ], "labPIs" : [ "Martin Zoltner" ], "affiliations" : [ "Head of Drug Discovery and Evaluation, Charles University in Prague, Department of Parasitology, BIOCEV, Průmyslová 595, 252 50 Vestec, Czech Republic" ], "instruments" : [ "Orbitrap Fusion" ], "softwares" : [ ], "quantificationMethods" : [ "label-free quantification" ], "sampleAttributes" : [ "cell culture", "permanent cell line cell", "Trypanosoma brucei", "Trypanosomiasis" ], "organisms" : [ "Trypanosoma brucei" ], "organismsPart" : [ "Cell culture", "Permanent cell line cell" ], "diseases" : [ "Trypanosomiasis" ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "BioID_PCF_wtcontrol_3.raw", "BiP_PCF_1.raw", "BiPN_PCF_3.raw", "BioID_BSF_wtcontrol_2.raw", "BIP_BSF_1.raw", "BiPN_BSF_2.raw", "BIP_BSF_3.raw", "BIP_PCF_2.raw", "BiPN_PCF_1.raw", "NUP65C_2.raw", "BiP_combined.txt.rar", "BioID_PCF_wtcontrol_1.raw", "NUP65C_1.raw", "BioID_BSF_wtcontrol_1.raw", "checksum.txt", "NUP65C_combined.txt.rar", "BiPN_BSF_3.raw", "TriTrypDB-64_TbruceiTREU927_AnnotatedProteins.fasta", "BiPN_BSF_1.raw", "BIP_BSF_2.raw", "BioID_BSF_wtcontrol_3.raw", "BIP_PCF_3.raw", "BioID_PCF_wtcontrol_2.raw", "BiPN_PCF_2.raw" ], "highlights" : { } }, { "accession" : "PXD075438", "title" : "Glycoproteomics of Human and Mouse Brains in Alzheimer’s Disease", "projectDescription" : "This study profiles glycoproteomic changes in human frontal cortex and mouse brain in Alzheimer’s disease models. We compare cognitively normal and AD human brain tissue, and wild-type and 5xFAD mouse brain, to define whether AD-associated hyperglycosylation reflects increased glycosylation of existing proteins. The work is integrated with spatial glycomics and related multiomic analyses to identify disease-associated glycopeptides and glycoproteins linked to neurodegeneration.", "dataProcessingProtocol" : "Raw glycoproteomics data were processed in Proteome Discoverer v3.0. Peptides were identified with CHIMERYS, and glycopeptides were assigned using the Byonic node. Search parameters included 5 ppm precursor tolerance and 10 ppm fragment tolerance. Variable modifications included deamidation, oxidation, and carboxymethylation. Glycopeptide identifications were filtered at log probability >1 and ppm error <3. Human samples were searched against the “N-glycan 182 human no multiple fucose” library, and mouse samples against the “N-glycan 309 mammalian no sodium” library. O-glycan searches used the “9-Most-common glycans” library. Curated outputs were used for relative glycopeptide quantification and downstream interpretation.", "sampleProcessingProtocol" : "De-identified human frontal cortex specimens and mouse brain tissues were collected and prepared under approved protocols. For glycoproteomics, pooled samples were used to ensure sufficient membrane protein input. Membrane proteins were enriched by high-salt extraction, sodium carbonate fractionation, and sequential ultracentrifugation. The membrane fraction was denatured in urea buffer, reduced, alkylated with iodoacetamide, precipitated by chloroform/methanol/water extraction, and resuspended in ammonium bicarbonate. Protein concentration was measured by BCA assay, and 100 µg of membrane protein was digested with trypsin at 37°C for 24 h. Glycopeptides were then analyzed by nanoLC on an Ultimate 3000 RSLCnano coupled to a Thermo Eclipse mass spectrometer using a 15 cm × 75 µm C18 column and a 180 min gradient.", "projectTags" : [ ], "keywords" : [ "Alzheimer’s disease; glycoproteomics; n-glycosylation; human brain; mouse brain" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-31", "updatedDate" : "2026-03-10", "submissionDate" : "2026-03-10", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "RAMON SUN" ], "labPIs" : [ "Ramon sun" ], "affiliations" : [ "university of florida" ], "instruments" : [ "LTQ Orbitrap" ], "softwares" : [ ], "quantificationMethods" : [ "Peptide counting" ], "sampleAttributes" : [ "Homo sapiens (Human)", "brain", "Alzheimer's disease" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Brain" ], "diseases" : [ "Alzheimer's disease" ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "AD_H_0_5_ug_ETD.raw", "Ngly_human.xlsx", "control_H_0_5_ug_ETD.raw", "control_M_0_5_ug_ETD.raw", "Ngly_mouse.xlsx", "AD_M_0_5_ug_ETD.raw" ], "highlights" : { } }, { "accession" : "PXD075464", "title" : "A novel player in trypanosome endoplasmic reticulum quality control with apparent similarities to the mammalian BiP nucleotide exchange inhibitor MANF", "projectDescription" : "We have identified the saponin-domain containing protein Tb927.5.1160 as a novel endoplasmic reticulum (ER) factor in Trypanosoma brucei. Tb927.5.1160 acts proximal to the prototypic ER chaperon BiP, the conserved heat shock protein 70 family chaperon playing a key role in protein folding and ER quality control, and exhibits a conserved BiP binding region shared with the mammalian BiP nucleotide exchange inhibitor MANF (mesencephalic astrocyte-derived neurotrophic factor). We demonstrated by inducible RNAi depletion and over-expression that Tb927.5.1160 has a marked effect on the sensitivity toward ER-stress and is essential for T. brucei viability. Here we deposit whole cell proteomics data monitoring protein abundance changes after 48 hours induction of Tb927.5.1160 RNAi in bloodstream form (BSF) and procyclic-form (PCF) T. brucei, and Tb927.5.1160 overexpression in BSF.", "dataProcessingProtocol" : "All data were analyzed and quantified with the Spectronaut 19 software (PMID: 25724911) using directDIA analysis. A spectral library was generated based on the predicted protein sequences for T. brucei TREU927 sourced from TriTrypDB (version 64) (PMID: 36656904). Enzyme specificity was set as C-terminal to Arg and Lys, also allowing cleavage at proline bonds and a maximum of two missed cleavages. Carbamidomethylation of cysteines was set as fixed modification and N- terminal protein acetylation and methionine oxidation as variable modifications. FDR was set to 1 % for PSM, peptide and protein. Quantification was performed on MS2 level. Precursor PEP cutoff and precursor and protein cutoff was set to 0.01, protein PEP was set to 0.05.", "sampleProcessingProtocol" : "Tetracycline inducible RNAi cell lines in T. brucei BSF and PCF, were generated using the pRPaiSL (PMID: 1858891) or the p3666 (PMID: 22511983) based stem-loop RNAi system, respectively, using primers predicted by RNAit2 (PMID: 12706807). Tb927.5.1160 inducible expression in T. brucei BSF relied on the plasmid construct pRPaTag and its integration into the tetracycline-responsive RRNA promoter of 2T1 cells (PMID: 16182389). PCF or BSF cells were induced with tetracyclin (1 g/ml) for Tb927.5.1160 RNAi depletion or overexpression and incubated for 48 h in parallel with nontreated cells. Cultures were harvested within their logarithmic growth phase and 5 x 107 cells resuspended in PBS containing Complete Mini Protease Inhibitor Mixture (Roche), then washed twice in PBS. Samples were then mixed with 100mM TEAB (Triethylammonium bicarbonate) containing 2% SDC (sodium deoxycholate) and lysed by boiling at 95°C for 5 min and sonication (Bandelin Sonoplus Mini 20, MS 1.5). Protein concentration was determined using BCA protein assay kit (Sigma Aldrich). 20 µg of protein was adjusted with 100mM TEAB (Triethylammonium bicarbonate) containing 2% SDC (sodium deoxycholate) to a final concentration of 50 µl, mixed with 40 mM chloroacetamide, 10mM TCEP (Tris(2-carboxyethyl)phosphine) and boiled again. Samples were then mixed with 5 µl of SP3 beads (PMID: 30464214) and 65 µl of 100 % ethanol. After binding, the beads were washed twice with 80% ethanol. The mixture was digested in 100 mM TEAB with 0.5 µg of trypsin overnight at 37°C. After digestion, samples were acidified with TFA to 1% final concentration and peptides were desalted using in-house made stage tips packed with C18 disks (Empore). Nano Reversed phase columns (Ion Opticks, Aurora Ultimate TS 25×75 C18 UHPLC column) were used for LC/MS analysis. LC was performed on a Vanquish NEO UHPLC system (Thermo Scientific). Mobile phase buffer A was composed of water and 0.1% formic acid. Mobile phase B was composed of acetonitrile and 0.1% formic acid. Samples were loaded onto the trap column (C18 PepMap100, 5 μm particle size, 300 μm x 5 mm, Thermo Scientific) in loading buffer composed of water and 0.1% formic acid. Peptides were eluted with a Mobile phase B gradient from 2% to 35% B in 9.6 min. Eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a Thermo Orbitrap Astral (Thermo Scientific) by data independent approach. Survey scans of peptide precursors from 380 to 980 m/z were performed in orbitrap at 120K resolution (at 200 m/z) with a 5 × 106 ion count target. DIA scans were performed in astral analyzer. AGC target was set to 5 × 104 and maximum injection time to 2 ms. Precursor mass range 380 – 980 m/z was covered by 4 m/z windows. Activation type was set to HCD with 25 % collision energy. Sample layout: (1)Tb927.5.1160 RNAi in PCF T. brucei 2 induced samples and 2 uninduced controls (Experiment 1847) (2)Tb927.5.1160 over-expression in BSF T. brucei 2 induced samples and 2 uninduced controls (Experiment 1914) (3)Tb927.5.1160 RNAi in BCF T. brucei 2 induced samples and 2 uninduced controls (Experiment 1922)", "projectTags" : [ ], "keywords" : [ "Trypanosoma bucei", "Endoplasmic reticulum", "Erqc" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-05", "updatedDate" : "2026-03-10", "submissionDate" : "2026-03-10", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Martin Zoltner" ], "labPIs" : [ "Martin Zolther" ], "affiliations" : [ "Department of Parasitology, Faculty of Science, Charles University in Prague, Biocev, Vestec, Prague, Czech Republic" ], "instruments" : [ "Orbitrap Astral" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "cell culture", "permanent cell line cell", "Trypanosoma brucei", "Trypanosomiasis" ], "organisms" : [ "Trypanosoma brucei" ], "organismsPart" : [ "Cell culture", "Permanent cell line cell" ], "diseases" : [ "Trypanosomiasis" ], "references" : [ ], "experimentTypes" : [ "Data-independent acquisition" ], "sdrf" : "", "projectFileNames" : [ "1914-BSFoverexpression-induced_replicate1.raw", "1914-BSFoverexpression-uninduced_control_replicate2.raw", "1847-PCFRNAi-uninduced_control-replicate1.raw", "1922-BSFRNAi.sne", "1922-BSFRNAi-uninduced_control-replicate2.raw", "1847-PCFRNAi-induced_replicate1.raw", "1914-BSFoverexpression-induced_replicate2.raw", "PCFRNAi_1847.sne", "1922-BSFRNAi-induced_replicate2.raw", "checksum.txt", "1914-BSFoverexpression-uninduced_control_replicate1.raw", "TriTrypDB-64_TbruceiTREU927_AnnotatedProteins.fasta", "1922-BSFRNAi-uninduced_control-replicate1.raw", "1847-PCFRNAi-uninduced_control-replicate2.raw", "1914-BSFoverexpression.sne", "1847-PCFRNAi-induced_replicate2.raw", "1922-BSFRNAi-induced_replicate1.raw" ], "highlights" : { } }, { "accession" : "PXD075393", "title" : "Proteomic profiling of conditioned medium from rotational trophoblast organoids (hTOr)", "projectDescription" : "Proteomic profiling of hTOr-conditioned medium was performed to characterize the secretome of rotational trophoblast organoids derived from human trophoblast stem cells. Collected supernatants underwent albumin depletion, followed by protein digestion using a Lys-C/trypsin mixture. The resulting peptides were analyzed by nanoLC-MS/MS using a Q Exactive mass spectrometer operating in data-independent acquisition (DIA) mode. This dataset is associated with a manuscript describing a rotational trophoblast organoid platform for modeling placental morphogenesis and tissue interactions.", "dataProcessingProtocol" : "Raw data were processed using DIA-NN software (v1.8.1) against the UniProtKB Human reference database with a peptide and protein false discovery rate (FDR) of less than 1 percent.", "sampleProcessingProtocol" : "Conditioned medium from rotational trophoblast organoids (hTOr) was collected at day 11 of culture. Supernatants underwent albumin depletion, followed by protein digestion using a Lys-C/trypsin mixture. The resulting peptides were desalted and prepared for nanoLC-MS/MS analysis.", "projectTags" : [ ], "keywords" : [ "Placenta", "Proteomics", "Secretome", "Human trophoblast organoids", "Dia" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-10", "updatedDate" : "2026-03-09", "submissionDate" : "2026-03-09", "downloadCount" : 35, "avgDownloadsPerFile" : 5.8333335, "botCount" : 9, "hubCount" : 26, "organicCount" : 0, "percentile" : 2, "yearlyDownloads" : [ { "year" : "2026", "count" : 35 } ], "submitters" : [ "Shun Shibata" ], "labPIs" : [ "Shun Shibata" ], "affiliations" : [ "Tohoku University Graduate School of Medicine" ], "instruments" : [ "Q Exactive" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "testis", "trophoblast cell" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Testis", "Trophoblast cell" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Shotgun proteomics" ], "sdrf" : "", "projectFileNames" : [ "240827_H56860_0668A_CT27_DIA_02.raw", "250116_H57819_0725A_B31_DIA_2.raw", "668A_tohokuU_240827_result.pg_matrix.tsv", "H57819_0725A_Normalaze_off_result.pg_matrix.tsv", "H57819_0725A_Normalaze_off_result.pr_matrix.tsv", "668A_tohokuU_240827_result.pr_matrix.tsv" ], "highlights" : { } }, { "accession" : "PXD075396", "title" : "E.coli total RNA RNase T1 and A digests LC-MS/MS", "projectDescription" : "Four open-source software suites for the analysis of oligonucleotide data from tandem MS have been reported. These tools are all based on matching theoretical spectral libraries generated by silico digestion. While some software publications have included simple comparisons with previously published tools, a discussion of oligonucleotide identification within complex backgrounds is lacking. In this study, we acquired multiple complex oligonucleotide datasets using an Orbitrap instrument to evaluate various search engines (Ariadne, RNAModMapper, NASE and Pytheas) and assess their performance in terms of accuracy, false discovery rate (FDR), performance at certain FDR, consensus of oligonucleotide identification, consistency of outputs in chemical measurements, and unknown modification discovery.In this study, we prepared two sets of samples, which involve three RNase T1 digested and three RNase A digested E. coli total RNA. Then, digests were analyzed by LC-MS/MS.", "dataProcessingProtocol" : "Ariadne, RNAModMapper (RAMM), NucleicAcidSearchEngine (NASE) and Pytheas were involved in the evaluation. The raw LC-MS/MS files were converted to either mgf or mzML format following software’s requirement. For software parameters’ settings, mass error was uniformed, which was set as 10 ppm (Δ 0.01 m/z) for precursor ions m/z and 15 ppm (Δ 0.05 m/z) for fragment ions m/z. In silico enzyme digestion miss cleavage was set to 2 for all applied samples.", "sampleProcessingProtocol" : "E. coli DH5α strain grown at 37 °C in the LB medium. Cells were precipitated by centrifugation and washed with PBS. 1 mL TRIzol was added to the E. coli and freeze under -80°C .TRIzol extraction method was applied to total RNA extraction. In brief, E. coli cells were lysed by adding TRIzol reagent at a ratio of 1:10 (w/v: mg/µL), followed by thorough mixing. The mixture was incubated on ice for 5 minutes, after which chloroform (one-fifth of the volume of TRIzol reagent used) was added and mixed vigorously. The sample was then centrifuged at 12,000 × g and 4 °C for 10 minutes. The upper aqueous phase was carefully transferred to a new tube, and isopropanol (equivalent to 0.7 volumes of the aqueous phase) was added. After thorough mixing, the sample was allowed to stand for 10 minutes and then centrifuged at 12,000 × g and 4 °C for 10 minutes to pellet the RNA. The RNA pellet was washed once with 75% ethanol, the supernatant was removed, and the pellet was air-dried briefly. Finally, the RNA was dissolved in RNase-free water. RNAs were heat-denatured for 5 min and digested by RNase T1 or A for 1 hour at 37 °C in RNase T1 buffer (100 mM Tris-HCl, pH 7.5 and 1mM EDTA) or RNase A buffer (30 mM ammonium acetate, pH 7.0). Digests were then heat-denatured for enzyme inactivation and 20 μg of each sample was used for single injection. Although identical RNA samples were used, different enzymatic digestion generated completely distinct oligonucleotide mixtures.", "projectTags" : [ ], "keywords" : [ "E.coli rna", "Lc-ms/ms", "Oligonucleotide" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-09", "updatedDate" : "2026-03-09", "submissionDate" : "2026-03-09", "downloadCount" : 27, "avgDownloadsPerFile" : 3.0, "botCount" : 25, "hubCount" : 2, "organicCount" : 0, "percentile" : 2, "yearlyDownloads" : [ { "year" : "2026", "count" : 27 } ], "submitters" : [ "Jing Feng" ], "labPIs" : [ "Xiang Zhang" ], "affiliations" : [ "University of Louisville, Department of Chemistry, Louisville, KY 40208, USA" ], "instruments" : [ "Orbitrap Fusion Lumos" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "cell culture", "Escherichia coli" ], "organisms" : [ "Escherichia coli" ], "organismsPart" : [ "Cell culture" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "23HCD_2_A.mzML", "23HCD_3_A.mzML", "23HCD_1_A.mzML", "Ecoli_rRNA.fasta", "23HCD_1.mzML", "23HCD_3.mzML", "RNase_Digests_score.csv", "23HCD_2.mzML" ], "highlights" : { } }, { "accession" : "PXD075406", "title" : "Proteomic analysis of small intestine luminal contents in IL22Fc-treated mouse", "projectDescription" : "Proteomic analyis was perfomred to identify the IL22Fc-induced proteins/polypeptides in the murine small intestine contents", "dataProcessingProtocol" : "MS raw files were searched against the UniProt FASTA databases using Sequest (Thermo Fisher Scientific). Databases included reversed (decoy) sequences to enable estimation of false discovery rates. Peptide-spectrum matches were filtered to achieve a peptide-level false discovery rate of 1–2%. Carbamidomethylation of cysteine residues was specified as a fixed modification during database searching. Enzyme specificity was defined as cleavage at the carboxyl terminus of arginine and lysine residues, consistent with digestion by trypsin and LysC. A maximum of 1 missed cleavages was allowed. All bioinformatic analyses were performed using Perseus (version 2.0.7.0) as well as standard analytical workflows implemented in R (version 4.5.2).", "sampleProcessingProtocol" : "The distal SI tissue was collected from control or IL22Fc-treated infant animals and cut open longitudinally. The tissue was incubated in 4 mL of wash buffer [1mM DTT PBS with protease inhibitor (Roche, 4693159001)] on ice for 10 min and vortexed for 20 seconds. After the removal of tissue pieces, intestinal washes were centrifuged at 100 xg for 1 min to remove the large pieces of intestinal contents. The supernatant was concentrated by centrifugation at 4000 xg with 3 kDa MWCO Amicon Ultra centrifugal filter unit for 1 hour. The volume of the concentrate was adjusted to 400 mL with wash buffer. The supernatants were processed for LC-MS/MS analysis using the ENRICH-iST kit (PreOmics, #P.O.00163). Assay details have been described previously, including protein enrichment, denaturation, reduction, alkylation, enzymatic digestion, and peptide cleanup within a single workflow. Briefly, mucus proteins were captured on paramagnetic enrichment beads in binding buffer, followed by multiple bead-based wash steps to remove salts, lipids, and low-molecular-weight contaminants. Bead-captured proteins were denatured, reduced, and alkylated using the supplied lysis buffer at elevated temperature and digested on-bead with a Trypsin/LysC protease mixture. Digestion was terminated by stop solution, and peptides were purified using the solid-phase extraction cartridge with sequential washes to remove impurities. Peptides were eluted in volatile buffer, dried completely by vacuum centrifugation (SpeedVac SPD120), and stored at −80 °C until LC-MS/MS analysis.Peptides were reconstituted in 5-10 µL of solvent A (2.5% acetonitrile, 0.1% formic acid) and separated by nano-scale reverse-phase liquid chromatography using a custom-packed capillary column (100 µm inner diameter × ~30 cm length) containing 2.6 µm C18 spherical silica beads and terminated with a flame-drawn electrospray tip. Subsequently, peptides were loaded via an autosampler and eluted using a linear gradient of increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid). Eluting peptides were ionized by electrospray and analyzed on an Orbitrap Velos Pro hybrid ion trap-Orbitrap mass spectrometer (Thermo Fisher Scientific).", "projectTags" : [ ], "keywords" : [ "Luminal contents", "Mouse", "Il22fc", "Small intestine" ], "doi" : "10.6019/PXD075406", "submissionType" : "COMPLETE", "publicationDate" : "2026-04-13", "updatedDate" : "2026-03-09", "submissionDate" : "2026-03-09", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Zhu Liang" ], "labPIs" : [ "Matthew K Waldor" ], "affiliations" : [ "1.Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA, USA 2.Department of Microbiology, Harvard Medical School, Boston, MA, USA 3.Howard Hughes Medical Institute, Boston, MA, USA" ], "instruments" : [ "LTQ Orbitrap Velos" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Mus musculus (Mouse)", "small intestine mucosa" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Small intestine mucosa" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition", "Top-down proteomics" ], "sdrf" : "", "projectFileNames" : [ "118325.raw", "112708_118330.mzid", "118330.mzXML", "118325.mzXML", "118329.raw", "checksum.txt", "118323.mzXML", "112707_118331.mzid", "118328.mzXML", "112710_118328.mzid", "118324.raw", "112688_118322.mzid", "118328.raw", "112709_118329.mzid", "112714_118324.mzid", "118322.raw", "118331.raw", "112711_118327.mzid", "118326.mzXML", "118323.raw", "118324.mzXML", "118331.mzXML", "118327.raw", "118326.raw", "118329.mzXML", "118322.mzXML", "112712_118326.mzid", "112689_118323.mzid", "112713_118325.mzid", "118330.raw", "118327.mzXML" ], "highlights" : { } }, { "accession" : "PXD075407", "title" : "Proteomics of Mouse Testicular Organoids on Day 6", "projectDescription" : "To investigate the mechanism for the formation of tubule-like structures in testicular organoids, we conducted proteomic analysis on O-Torgs and T-Torgs at day 6. Through differential protein expression and enrichment analysis, it was found that the extracellular matrix (ECM) related pathways in O-Orgs were significantly upregulated. This indicates that the ECM might facilitate the formation of tubule-like structures in O-Orgs, which might be important for spermatogenesis.", "dataProcessingProtocol" : "Raw DIA data were processed with Spectronaut 19 against the 2024 mouse UniProt database, accounting for variable and fixed modifications. Dynamic iRT was used for retention time prediction, and Q value cutoffs were set at 1% for precursors, peptides, and proteins. Decoy generation was mutated, and local normalization was applied. The MaxLFQ method was used to determine major group quantities from peptides exceeding the 1% Q value threshold. Differential proteins were identified by comparing the fold change of the biological replicate means between groups.", "sampleProcessingProtocol" : "Protein extraction and tryptic digestion of day 6 testicular organoids were performed with the iST kit. Each replicate consists of approximately 200 organoids and each group has three independent biological replicates. After heating and cooling, samples were incubated, then cleaned and desalted. Peptides were separated on a Vanquish Neo UHPLC system with a C18 column, and DIA data was collected in diaPASEF mode using a Bruker timsTOF HT mass spectrometer.", "projectTags" : [ ], "keywords" : [ "Lc-msms", "Mouse", "Testicular organoids" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-26", "updatedDate" : "2026-03-09", "submissionDate" : "2026-03-09", "downloadCount" : 6, "avgDownloadsPerFile" : 0.46153846, "botCount" : 0, "hubCount" : 6, "organicCount" : 0, "percentile" : 1, "yearlyDownloads" : [ { "year" : "2026", "count" : 6 } ], "submitters" : [ "Qi Li" ], "labPIs" : [ "Xiao-Yang Zhao" ], "affiliations" : [ "State Key Laboratory of Organ Failure Research, School of Laboratory Medicine and Biotechnology, Southern Medical University" ], "instruments" : [ "timsTOF HT" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "testis", "Mus musculus (Mouse)" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Testis" ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.1038/S41467-026-71254-W" ], "experimentTypes" : [ "SWATH MS" ], "sdrf" : "", "projectFileNames" : [ "O-Torgs_Day6_rep2.d.zip", "T-Torgs_Day6_rep3.d.zip", "Spectronaut_T-Torgs_Day6_rep2.m.zip", "Spectronaut_O-Torgs_Day6_rep2.m.zip", "Spectronaut_O-Torgs_Day6_rep3.m.zip", "O-Torgs_Day6_rep1.d.zip", "T-Torgs_Day6_rep2.d.zip", "Spectronaut_T-Torgs_Day6_rep3.m.zip", "T-Torgs_Day6_rep1.d.zip", "Spectronaut_O-Torgs_Day6_rep1.m.zip", "checksum.txt", "O-Torgs_Day6_rep3.d.zip", "Spectronaut_T-Torgs_Day6_rep1.m.zip" ], "highlights" : { } }, { "accession" : "PXD075355", "title" : "Distinct physiological strategies underpin antagonism of native Pichia yeasts against Botrytis cinerea", "projectDescription" : "Proteomics dataset generated using LC-MS/MS on a Bruker timsTOF instrument with DDA-PASEF acquisition. Protein identification was performed using PEAKS software. The dataset includes raw instrument files (.d folders) and processed peptide and protein identification results.", "dataProcessingProtocol" : "Raw LC–TIMS–MS/MS data were processed using PEAKS Studio ProX (Bioinformatics Solutions Inc.). MS/MS spectra were searched against a combined UniProt database containing the reference proteomes of the analysed yeast strains and Botrytis cinerea. Search parameters included a precursor mass tolerance of 15 ppm and fragment tolerance of 0.05 Da, with carbamidomethylation of cysteine as fixed modification and methionine oxidation and N-terminal acetylation as variable modifications. Protein identifications were validated using a target–decoy strategy with a false discovery rate (FDR) threshold of 1%. Proteins supported by at least two unique peptides and −10lgP ≥ 25 were retained for downstream analysis. Protein intensities were log2-transformed, normalized by median centering, and missing values were imputed using a minimum-intensity approach. Differential abundance analysis was performed using empirical Bayes linear modelling implemented in the limma package in R (v4.3.2), applying Benjamini–Hochberg correction and considering proteins with FDR ≤ 0.05 and |log2FC| ≥ 0.58 as differentially abundant.", "sampleProcessingProtocol" : "Protein samples were digested into peptides and analysed by nanoLC–TIMS–MS/MS. Peptide mixtures (200 ng per injection) were separated using a nanoElute UHPLC system (Bruker Daltonics) coupled to a timsTOF Pro 2 mass spectrometer equipped with a CaptiveSpray nanoelectrospray source. Peptides were loaded onto a Bruker FIFTEEN C18 column (15 cm × 75 μm ID, 1.9 μm particle size) and separated at 30 °C using a 20-min gradient at 300 nL min⁻¹ (mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid in acetonitrile). The elution gradient was 0–35% B for 13 min, 35–90% B from 13–15 min, followed by a wash at 90% B. Mass spectrometry acquisition was performed on a timsTOF Pro 2 in data-dependent acquisition mode using the PASEF method, acquiring MS and MS/MS spectra in the m/z range 100–1700. Ion mobility separation ranged from 0.85 to 1.30 V·s cm⁻² with a ramp time of 100 ms. Four PASEF MS/MS scans were acquired per cycle. Active exclusion was set to 0.4 min for precursors above 20,000 intensity units.", "projectTags" : [ ], "keywords" : [ "Proteomics lc-ms/ms dda-pasef peaks biocontro" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-21", "updatedDate" : "2026-03-07", "submissionDate" : "2026-03-07", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Jose Padilla" ], "labPIs" : [ "Jose Luis Padilla Agudelo" ], "affiliations" : [ "Department of Biomedicine, Biotechnology and Public Health, University of Cádiz, Spain" ], "instruments" : [ "timsTOF Pro" ], "softwares" : [ ], "quantificationMethods" : [ "label-free quantification" ], "sampleAttributes" : [ "Pichia kluyveri", "cell culture", "vegetative cell (sensu Fungi)", "Pichia kudriavzevii" ], "organisms" : [ "Pichia kluyveri", "Pichia kudriavzevii" ], "organismsPart" : [ "Cell culture", "Vegetative cell (sensu fungi)" ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.1016/J.FM.2026.105112" ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "JesusCantoral_sol791-34-25_14_CA4_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-14_1_9-12-2025_8337.d.zip", "de_novo_only_peptides.csv", "JesusCantoral_sol791-34-25_2_B05.10_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-2_1_9-11-2025_8298.d.zip", "peptides.html", "JesusCantoral_sol791-34-25_8_BF8_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-8_1_9-12-2025_8319.d.zip", "proteins.csv", "protein-peptides.csv", "JesusCantoral_sol791-34-25_7_BF8_B05.10_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-7_1_9-12-2025_8313.d.zip", "JesusCantoral_sol791-34-25_9_BF8_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-9_1_9-12-2025_8322.d.zip", "JesusCantoral_sol791-34-25_12_CA4_B05.10_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-12_1_9-12-2025_8331.d.zip", "protein.html", "JesusCantoral_sol791-34-25_1__NegCont_PDB_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-1_1_9-11-2025_8296.d.zip", "JesusCantoral_sol791-34-25_6_BF8_B05.10_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-6_1_9-11-2025_8310.d.zip", "JesusCantoral_sol791-34-25_11_CA4_B05.10_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-11_1_9-12-2025_8328.d.zip", "peptide.csv", "checksum.txt", "JesusCantoral_sol791-34-25_10_BF8_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-10_1_9-12-2025_8325.d.zip", "JesusCantoral_sol791-34-25_15_CA4_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-15_1_9-12-2025_8340.d.zip", "JesusCantoral_sol791-34-25_5_BF8_B05.10_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-5_1_9-11-2025_8307.d.zip", "JesusCantoral_sol791-34-25_13_CA4_B05.10_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-13_1_9-12-2025_8334.d.zip", "JesusCantoral_sol791-34-25_16_CA4_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-16_1_9-12-2025_8343.d.zip", "JesusCantoral_sol791-34-25_4_B05.10_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-4_1_9-11-2025_8304.d.zip", "JesusCantoral_sol791-34-25_3_B05.10_DDA-PASEF_SG_0.8-1.5_Aurora25cm_30min_Slot2-3_1_9-11-2025_8301.d.zip", "DB_search_psm.csv" ], "highlights" : { } }, { "accession" : "PXD075335", "title" : "The environmental stress response regulates biophysics of the cytoplasm and survival in quiescence", "projectDescription" : "All organisms have evolved survival strategies to cope with changes in environmental conditions. Nutrient deprivation, one of the most frequently encountered stresses in nature, causes haploid budding yeast to enter a reversible state of non-proliferation known as quiescence, which entails extensive remodeling of gene expression, metabolism and the cellular biophysical properties. Yeast cells can adapt to and survive long periods of time in glucose starvation-induced quiescence, provided they are able to respire in the early stages of glucose withdrawal. When respiration is blocked during glucose withdrawal, cells prematurely age and exhibit markedly reduced survival and cytoplasmic diffusion. We find here that respiration is required to induce a quiescence-related gene expression program. Induction of this program prior to withdrawing glucose in respiration-inhibited cells bypasses the need for respiration and rescues survival and biophysical properties to levels seen in glucose-starved but respiration-competent cells. This rescue effect relies on proteomic adaptation, which partially occurs through inactivation of Ras/PKA signaling and activation of the environmental stress response via the transcription factors Msn2/4. This signaling cascade triggers the expression of stress response genes and modulates the cytoplasmic diffusion state of cells, ensuring long-term survival in quiescence even in the absence of respiration. Our results highlight the importance of stress adaptation in quiescence and aging, integrating gene expression control and modulation of cytoplasmic properties to maintain cell fitness.", "dataProcessingProtocol" : "DIA data were analyzed in Spectronaut (v18.6) using the directDIA workflow. Spectra were searched against the Saccharomyces Genome Database protein database (6713 entries) with carbamidomethylation as a fixed modification, and methionine oxidation and N-terminal protein acetylation as a variable modification. Peptide and protein identifications were filtered at 1% false discovery rate. Fragment ion intensities were exported and further processed in R, retaining only proteotypic precursors and summing fragment ions to precursor intensities. Protein quantification was performed using the MaxLFQ algorithm. Proteins supported by at least five precursors in four replicates of at least one condition were retained. Missing values were imputed from a distribution representing low-intensity signals, followed by median normalization across samples. Statistical significance between conditions was assessed using two-sided Student’s t-tests with Benjamini–Hochberg correction for multiple testing.", "sampleProcessingProtocol" : "Yeast cells were grown exponentially in SCD medium and either left untreated or subjected to heat shock (42 °C for 30 min followed by 30 °C for 30 min). Cultures were washed into SC medium with or without antimycin A and incubated at 30 °C for 1 h or 20 h before harvesting by centrifugation. Cell pellets were lysed in HEPES-based lysis buffer containing protease inhibitors using glass bead beating at 4 °C. Protein concentrations were determined by BCA assay, and equal amounts of protein were reduced with TCEP, alkylated with iodoacetamide, and digested with Lys-C and trypsin overnight at 37 °C. Peptides were acidified, desalted using C18 solid-phase extraction, dried, and resuspended prior to LC–MS/MS analysis. Peptide separation was performed by nanoflow LC and analyzed on an Orbitrap Exploris 480 mass spectrometer operating in a data-independent acquisition mode.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-09", "updatedDate" : "2026-03-06", "submissionDate" : "2026-03-06", "downloadCount" : 136, "avgDownloadsPerFile" : 3.090909, "botCount" : 0, "hubCount" : 136, "organicCount" : 0, "percentile" : 6, "yearlyDownloads" : [ { "year" : "2026", "count" : 136 } ], "submitters" : [ "Jonas S Fischer" ], "labPIs" : [ "Karsten Weis" ], "affiliations" : [ "Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich CH-8093, Switzerland" ], "instruments" : [ "Thermo Fisher Scientific instrument model" ], "softwares" : [ "Spectronaut" ], "quantificationMethods" : [ "Relative quantification" ], "sampleAttributes" : [ "Saccharomyces cerevisiae (Baker's yeast)" ], "organisms" : [ "Saccharomyces cerevisiae (baker's yeast)" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "SWATH MS" ], "sdrf" : "", "projectFileNames" : [ "200619_EX1_cdoerig_001_59.raw", "200619_EX1_cdoerig_001_54.raw", "200619_EX1_cdoerig_001_71.raw", "200619_EX1_cdoerig_001_46.raw", "2025-08-18_maxLFQdata.tsv", "200619_EX1_cdoerig_001_62.raw", "200619_EX1_cdoerig_001_37.raw", "200619_EX1_cdoerig_001_38.raw", "200619_EX1_cdoerig_001_68.raw", "200619_EX1_cdoerig_001_70.raw", "20240517_152807_2024-05-17_CarmenProteomics_directDIA_noPTM_wholeProteome_Report.tsv", "20240517_152807_2024-05-17_CarmenProteomics_directDIA_noPTM_wholeProteome_Report.log.txt", "200619_EX1_cdoerig_001_55.raw", "200619_EX1_cdoerig_001_40.raw", "200619_EX1_cdoerig_001_42.raw", "checksum.txt", "200619_EX1_cdoerig_001_47.raw", "200619_EX1_cdoerig_001_64.raw", "200619_EX1_cdoerig_001_39.raw", "200619_EX1_cdoerig_001_61.raw", "SampleAnnotationFile.xlsx", "200619_EX1_cdoerig_001_52.raw", "200619_EX1_cdoerig_001_43.raw", "200619_EX1_cdoerig_001_69.raw", "200619_EX1_cdoerig_001_56.raw", "200619_EX1_cdoerig_001_60.raw", "200619_EX1_cdoerig_001_48.raw", "200619_EX1_cdoerig_001_65.raw", "200619_EX1_cdoerig_001_51.raw", "200619_EX1_cdoerig_001_44.raw", "200619_EX1_cdoerig_001_57.raw", "200619_EX1_cdoerig_001_53.raw", "200619_EX1_cdoerig_001_72.raw", "200619_EX1_cdoerig_001_66.raw", "20240517_152807_2024-05-17_CarmenProteomics_directDIA_noPTM_wholeProteome_Report.params", "200619_EX1_cdoerig_001_49.raw", "lgilletSGD13Jan2015iRTZZCON.fasta", "200619_EX1_cdoerig_001_50.raw", "200619_EX1_cdoerig_001_63.raw", "200619_EX1_cdoerig_001_45.raw", "200619_EX1_cdoerig_001_67.raw", "200619_EX1_cdoerig_001_58.raw", "20240517_152807_2024-05-17_CarmenProteomics_directDIA_noPTM_wholeProteome_Report.setup.txt", "200619_EX1_cdoerig_001_41.raw" ], "highlights" : { } }, { "accession" : "PXD075330", "title" : "putzig safeguards genome integrity by contributing to the Piwi-mediated repression of transposon activity in the female germline of Drosophila in a two-tiered fashion", "projectDescription" : "Genome integrity in the germline is jeopardized by the activity of transposable elements (TEs). Transposon activity is kept in check by the Piwi-piRNA pathway, which silences TEs post-transcriptionally in the cytoplasm as well as co-transcriptionally in the nucleus. piRNAs derive from long precursor transcripts originating at piRNA-loci by non-conventional transcription. They are processed in the cytoplasm and loaded into Piwi-piRNA complexes that then enter the nucleus to bind to the target RNA and direct local heterochromatin formation at TE loci, involving several downstream effectors. In this work, we have analyzed the role of Putzig (Pzg) in Piwi-mediated TE silencing in the female germline. Pzg has multi-facetted roles during Drosophila development and is important for DNA integrity, germ cell differentiation and survival. Here, we provide evidence for a two-tier activity of Pzg by serving as a hub for various Piwi-pathway members. Firstly, Pzg assists transcription initiation at piRNA clusters by coupling the Trf2-Moonshiner transcription initiation complex to the Rhino-Deadlock-Cutoff complex, thereby promoting formation of piRNA-precursor transcripts. Secondly, in the context of co-transcriptional gene silencing, Pzg licences heterochromatin formation by linking the Piwi-piRNA silencing machinery and the histone demethylase Lsd1, involved in promoter inactivation.", "dataProcessingProtocol" : "The spectra were initially evaluated in MaxQuant (2.6.7.0) and the integrated search engine Andromeda. The detected peptides were assigned to individual proteins or protein groups, which were then quantified based on the calculation of LFQ intensities. Unless otherwise specified, the MaxQuant parameters were left in default mode. The reference proteome of Drosophila (ID: UP000000803) was obtained on 8 October 2024 from https://www.uniprot.org (The UniProt Consortium et al., 2025). Only peptides with a maximum of two undigested trypsin cleavage sites and exclusively unmodified amino acids, with the exception of oxidation, carbamidomethylation and deamidation (N-terminal) modifications, were included. MS and MS/MS mass tolerance was set to 20 ppm. Peptides that could be assigned to either a single protein or multiple proteins (unique and razor) were quantified. In the latter case, a peptide was assigned to the protein that had the highest probability of actually being present in the sample. Only proteins with a false discovery rate (FDR) of 1% or less were evaluated. The proteins identified by MaxQuant were then filtered together with their calculated LFQ intensities in Perseus (2.0.11) and statistically evaluated. Proteins that were identified solely on the basis of one or more peptides with modified amino acids, potential contaminations and reverse sequences were filtered out. In addition, a log2 transformation of the LFQ intensities was performed. The three IP and BC replicates were grouped and only proteins identified in all IP replicates were retained. If intensity values for proteins identified in all IP samples were missing in the BC replicates, these were supplemented by normal distribution. Significant differences between the average LFQ intensities of the IP and BC approaches were determined using a Student's t-test.", "sampleProcessingProtocol" : "A transgenic fly strain with GFP-labelled Pzg protein was used to precipitate Pzg using anti-GFP nanobodies (GFP-Trap®). 200 ovaries were prepared in PBS and homogenized in 450 µl protein lysis buffer [20 mM TRIS-HCl pH 7.5, 150 mM NaCl, 2 mM MgCl2, 10 % glycerol, 1 mM DTT, 1 mM Pefabloc® SC-Protease Inhibitor, 0.2 % NP40 in ddH2O] in a glass homogenizer with a size B pestle. After centrifugation for 15 minutes at 4°C with 14.000 rpm, the lysate was transferred to a fresh Eppendorf tube. Precipitation was carried out using magnetic GFP-Trap® and Binding Control agarose beads were used as a negative control. 200 µl of protein extract and 300 µl of washing solution 1 [150 mM NaCl, 50 mM TRIS-HCl pH 7.5, 0.1 % SDS, 1 tablet of protease inhibitor cocktail in ddH2O] were added to 25 µl of agarose beads and the mixtures were gently rotated for 1 hour at 4°C. Agarose beads were washed once with protein lysis buffer, three times with wash solution 1 and twice with washing solution 2 [150 mM NaCl, 50 mM TRIS-HCl pH 7.5, in ddH2O], with the beads being transferred to a fresh Eppendorf tube in the last step. The proteins were eluted from the beads in 60 µl of 6 M urea and 50 mM Tris-HCl pH 8.5. The lysate was then processed directly to mass spectrometry. The analysis was performed by UHPLC-ESI-MS/MS. First, the peptides digested overnight with trypsin were separated according to size using ultra-high-performance liquid chromatography (UHPLC) in the UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) and ionised using electrospray (ESI). The signals were then recorded on the Orbitrap Exploris™ 480 mass spectrometer (Thermo Fisher Scientific) with subsequent Fourier transformation to determine the mass-to-charge ratio. Tandem mass spectrometry (MS/MS) was performed, in which MS1 spectra with total peptide masses were generated alongside MS2 spectra with the masses of fragmented peptides.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "10.6019/PXD075330", "submissionType" : "COMPLETE", "publicationDate" : "2026-03-11", "updatedDate" : "2026-03-06", "submissionDate" : "2026-03-06", "downloadCount" : 42, "avgDownloadsPerFile" : 2.1, "botCount" : 1, "hubCount" : 41, "organicCount" : 0, "percentile" : 3, "yearlyDownloads" : [ { "year" : "2026", "count" : 42 } ], "submitters" : [ "Lea Hüttinger" ], "labPIs" : [ "Anja Nagel" ], "affiliations" : [ "Department of Molecular Genetics (190g), University of Hohenheim" ], "instruments" : [ "Orbitrap Exploris 480", "Dionex UltiMate 3000 RSLCnano" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "ovary", "Drosophila melanogaster (Fruit fly)" ], "organisms" : [ "Drosophila melanogaster (fruit fly)" ], "organismsPart" : [ "Ovary" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "proteinGroups.txt", "peptides.txt", "mzTab.mzTab", "mqpar.xml", "IP2.raw", "mzRange.txt", "tables.pdf", "IP3.raw", "parameters.txt", "BC2.raw", "evidence.txt", "BC4.raw", "UP000000803_7227.fasta", "checksum.txt", "msms.txt", "BC3.raw", "IP4.raw", "msmsScans.txt", "summary.txt", "modificationSpecificPeptides.txt" ], "highlights" : { } }, { "accession" : "PXD075291", "title" : "Competitive activity-based protein profiling of LF-268 target engagement in live HCT-116 cells using a click handle-derivatized RAF inhibitor probe", "projectDescription" : "Activity-based protein profiling using 268-TCO, a trans-cyclooctene (TCO)-derivatized analogue of the pan-RAF DFG-out inhibitor LF-268, to characterize intracellular target engagement in live HCT-116 (mutant KRAS/WT RAF) cells. Cells were pre-treated with LF-268 (5 µM) or DMSO as competitor, followed by treatment with 268-TCO probe. After lysis, probe-bound protein complexes were captured via click chemistry using tetrazine (Tz)-coupled sepharose beads. Enriched proteins were identified and quantified by label-free LC-MS/MS.", "dataProcessingProtocol" : "Raw data analyzed with MaxQuant (Andromeda search engine) against UniProt human database (July 2015). Settings: multiplicity=1; enzyme=trypsin/P; variable modifications: oxidation (M), acetyl (protein N-term); LFQ minimum ratio count=2; LFQ minimum neighbors=3; LFQ average neighbors=6; MS/MS tolerance=10 ppm; FDR=0.01. Perseus downstream analysis: proteins classified as \"only identified by site,\" \"reverse,\" or \"contaminant\" excluded; proteins with ≤1 unique peptide excluded. LFQ intensities log2-transformed; missing values imputed from a downshifted normal distribution (shift=1.3, width=0.25). Enrichment scores calculated as log2[Intensity(268-TCO) / Intensity(DMSO)]; confidence values as −log10(p-value) from two-tailed two-sample t-test.", "sampleProcessingProtocol" : "HCT-116 cells grown in DMEM supplemented with 10% FBS. Treated with LF-268 (5 µM) or DMSO (0.1% v/v) for competitor pre-treatment, followed by addition of 268-TCO probe (5 µM) for 30 min at 37°C. Medium removed, cells washed, lysed with RIPA buffer (50 mM Tris-HCl pH 7.8, 120 mM NaCl, 10 mM NaF, 1 mM Na₃VO₄, 1 mM EDTA, 1% IGEPAL CA-630, protease inhibitors), cleared by centrifugation and quantified by Pierce 660 nm assay, then diluted to 1.5 mg/mL. Lysates pre-cleared with ethanolamine-coupled NHS-Sepharose beads (15 µL drained) for 1 h at 4°C. Pre-cleared lysate (750 µg) incubated with Tz-beads (15 µL drained, washed 3× with lysis buffer) for 30 min at 4°C with end-over-end rotation. Beads washed several times, denatured, and sequentially digested with Lys-C then Trypsin. Desalted on C18 StageTip, dried by vacuum centrifugation, reconstituted in buffer then run on LC-MS.", "projectTags" : [ ], "keywords" : [ "Human", "Dda", "Lc-msms", "Kinase" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-20", "updatedDate" : "2026-03-06", "submissionDate" : "2026-03-06", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Daniel Brush" ], "labPIs" : [ "Dustin James Maly" ], "affiliations" : [ "Department of Chemistry, University of Washington" ], "instruments" : [ "Orbitrap Fusion Lumos" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)", "colon" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Colon" ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.1038/S41589-026-02212-2" ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "2-112_Co-CP_EphA2_HCT-116_TCO_neg-4hrs_3.raw", "2-112_Co-CP_EphA2_HCT-116_TCO_withcompetitor-4hrs_1.raw", "checksum.txt", "2-112_Co-CP_EphA2_HCT-116_TCO_withcompetitor-4hrs_2.raw", "proteinGroups_MaxQuant.txt", "2-112_Co-CP_EphA2_HCT-116_TCO_withcompetitor-4hrs_3.raw", "2-112_Co-CP_EphA2_HCT-116_TCO_neg-4hrs_2.raw", "2-112_Co-CP_EphA2_HCT-116_TCO_neg-4hrs_1.raw", "peptides_MaxQuant.txt" ], "highlights" : { } }, { "accession" : "PXD075307", "title" : "Phosphoproteomic profiling of kinobead-enriched fractions from LF-268-treated HCT-116 cells via SILAC-based quantification", "projectDescription" : "SILAC-based phosphoproteomic profiling of kinase-enriched fractions from HCT-116 cells treated with the DFG-out pan-RAF inhibitor LF-268 to characterize kinase signaling responses. Cells were metabolically labeled and treated with 1 µM LF-268 or DMSO for 1 hour, followed by serial kinobead enrichment and Fe³⁺-NTA IMAC phosphopeptide enrichment prior to LC-MS/MS.", "dataProcessingProtocol" : "Raw data analyzed with MaxQuant (Andromeda search engine) against UniProt human database (July 2015). Settings: multiplicity=2; SILAC labels Arg0/Lys0 (light) and Arg10/Lys8 (heavy); enzyme=trypsin/P; variable modifications: oxidation (M), acetyl (protein N-term), phospho (STY); LFQ minimum ratio count=2; LFQ minimum neighbors=3; LFQ average neighbors=6; MS/MS tolerance=10 ppm; FDR=0.01. Perseus downstream analysis: proteins classified as \"only identified by site,\" \"reverse,\" or \"contaminant\" excluded; proteins with ≤1 unique peptide excluded. Intensities log2-transformed; missing values imputed from a downshifted normal distribution (shift=1.3, width=0.2). H/L ratios reported as log2[LF-268/DMSO]. Significantly regulated phosphosites defined by competition score ≥ 1.5 and confidence value (−log10 p-value) ≥ 1.5 from two-tailed two-sample t-test, FDR=0.05.", "sampleProcessingProtocol" : "HCT-116 cells grown in SILAC DMEM supplemented with 10% FBS, labeled with heavy (Arg10/Lys8) or light (Arg0/Lys0) isotopes. Treated with LF-268 (1 µM) or DMSO for 1 hour. Lysed with ice-cold modified RIPA buffer (50 mM Tris pH 7.8, 150 mM NaCl, 10 mM NaF, 1% IGEPAL CA-630, 0.25% sodium deoxycholate, 5% glycerol, protease inhibitors, 1 mM PMSF, phosphatase inhibitor cocktails 2 & 3) on 15-cm plates (250 µL), cleared at 17,000×g for 10 min at 4°C, and diluted to 2.0 mg/mL by Pierce 660 nm assay. Lysate (150 µL) incubated with kinobeads (10 µL 50% slurry, pre-washed 2× with modified RIPA) for 3 h at 4°C with end-over-end rotation. Beads washed 2× modified RIPA and 3× ice-cold TBS (50 mM Tris pH 7.8, 150 mM NaCl). On-bead denaturation in 25 µL denaturing buffer (6 M guanidinium chloride, 100 mM Tris pH 8.5, 5 mM TCEP, 10 mM CAM) at 95°C for 5 min. Diluted with 25 µL 100 mM TEAB, pH adjusted to 8–9 with 1 N NaOH. Sequential digestion: LysC (0.4 µg, 37°C, 2 h, 1400 rpm), diluted with 50 µL 100 mM TEAB, trypsin (0.4 µg, 37°C, overnight, 800 rpm). Quenched with Buffer A (0.1% TFA/5% acetonitrile, 1% formic acid), supernatant loaded onto in-house C18 column, washed with Buffer A (50 µL), eluted with Buffer B (80% acetonitrile/0.1% TFA, 50 µL), resuspended in Buffer A (20 µL). Phosphopeptide enrichment: peptides resuspended in 80% aq. acetonitrile/0.1% TFA and incubated with Fe³⁺-NTA loaded beads (pre-washed 10× with 80% aq. acetonitrile/0.1% TFA). Eluted twice with 50 mM KH₂PO₄/NH₄OH (pH 10), combined, pH neutralized, dried, C18 cleanup, reconstituted in 80:20 ACN:water. n=3 biological replicates.", "projectTags" : [ ], "keywords" : [ "Human", "Dda", "Lc-msms", "Phosphoproteomics", "Kinase" ], "doi" : "", "otherOmicsLinks" : [ "pride.project:PXD075291", "pride.project:PXD075247" ], "submissionType" : "PARTIAL", "publicationDate" : "2026-04-20", "updatedDate" : "2026-03-06", "submissionDate" : "2026-03-06", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Daniel Brush" ], "labPIs" : [ "Dustin James Maly" ], "affiliations" : [ "Department of Chemistry, University of Washington" ], "instruments" : [ "Orbitrap Fusion Lumos" ], "softwares" : [ ], "quantificationMethods" : [ "SILAC" ], "sampleAttributes" : [ "Homo sapiens (Human)", "colon" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Colon" ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.1038/S41589-026-02212-2" ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "111718_HCT116_L1-1uM-LF-268_1hr_H1-DMSO_Phospho_tech2.raw", "111718_HCT116_L-DMSO_H-1uM-LF-268_1hr_Phospho_tech1.raw", "checksum.txt", "111718_HCT116_L2-1uM-LF-268_1hr_H2-DMSO_Phospho_tech1.raw", "111718_1uM_LF-268_1hr_phosphokinomics_w-Annotation.xlsx", "111718_HCT116_L2-1uM-LF-268_1hr_H2-DMSO_Phospho_tech2.raw", "111718_HCT116_L-DMSO_H-1uM-LF-268_1hr_Phospho_tech2.raw", "111718_HCT116_L1-1uM-LF-268_1hr_H1-DMSO_Phospho_tech1.raw" ], "highlights" : { } }, { "accession" : "PXD075275", "title" : "Global analysis of cancer cell responses to USP9X inhibition", "projectDescription" : "K-GG ubiquitin profiling of DLD-1 and MDA-MB-231 cells treated with the selective USP9X inhibitor WEHI-092 at 10 µM for 30 min, 6 h, and 24 h (and DMSO-treated control). This dataset also includes DLD-1 cells treated with FT671 (USP7 inhibitor) at 10 µM for 30 min and 6 h. Sample IDs: #1-5 Control 30 min DLD-1 #6-10 FT671 30 min DLD-1 #11-15 WEHI-092 30 min DLD-1 #16-20 Control 360 min DLD-1 #21-25 FT671 360 min DLD-1 #26-30 WEHI-092 360min DLD-1 #31-35 Control 30 min MDA-MB-231 #36-40 WEHI-092 30 min MDA-MB-231 #41-45 Control 360 min MDA-MB-231 #46-50 WEHI-092 360 min MDA-MB-231 #51-55 Control 24 h MDA-MB-231 #56-60 WEHI-092 24 h MDA-MB-231", "dataProcessingProtocol" : "MS raw files were processed using DIA-NN 1.8.1 as described previously (Steger et al, 2021). For K-GG experiments, library-free searching with K-GG variable modification enabled with a maximum of two modifications and two-missed cleavages was performed with the reviewed human proteome (Uniprot), with MBR enabled and Robust LC quantification. Precursors were consolidated by retaining the precursor that was detected in the largest number of samples and had the highest intensity. Data were filtered for K-GG peptides that were detected in four out of five samples in at least one condition (unless specified otherwise) and differential expression analysis performed using the DEP R package (Zhang et al, 2018). Data was normalised with variance stabilising normalisation (VSN) and missing values imputed using a mixed Bayesian principal component analysis (BPCA) and Min method (unless stated otherwise in the figure legends). Significance testing of log2-transformed intensities was performed, and Benjamini-Hochberg corrected p-values <0.05 were considered as significant. K-GG fold changes were corrected for changes in the proteome unless stated otherwise. Downstream processing and differential expression analysis was performed in R using limma (Ritchie et al, 2015) and DEP packages. For K-GG experiments, library-free searching with K-GG variable modification enabled with a maximum of two modifications and two-missed cleavages was performed with the reviewed human proteome (Uniprot), with MBR enabled and Robust LC quantification. Precursors were consolidated by retaining the precursor that was detected in the largest number of samples and had the highest intensity. Data were filtered for K-GG peptides that were detected in four out of five samples in at least one condition (unless specified otherwise) and differential expression analysis performed using the DEP R package (Zhang et al, 2018). Data was normalised with variance stabilising normalisation (VSN) and missing values imputed using a mixed Bayesian principal component analysis (BPCA) and Min method (unless stated otherwise in the figure legends). Significance testing of log2-transformed intensities was performed, and Benjamini-Hochberg corrected p-values <0.05 were considered as significant. K-GG fold changes were corrected for changes in the proteome unless stated otherwise.", "sampleProcessingProtocol" : "For global proteomics, cells were lysed in 200 µL of preheated (95°C) buffer (2.5% [v/v] SDS in 100 mM Tris-HCl, pH 8.5). DNA was hydrolysed with the addition of 2 µL neat TFA and lysates were neutralised to pH 8.5 by addition of 1 M Tris-HCl as previously described (Dagley et al, 2019). Protein concentration was determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. For global proteomics, cell lysates (20 µg protein per replicate) were transferred to 0.5 mL LoBind deep well plate (Eppendorf) prepared for MS analysis using the modified SP3 protocol (Hughes et al, 2019), with some modifications. Briefly, samples were subjected to simultaneous reduction and alkylation with a final concentration of 10 mM TCEP and 40 mM 2-chloracetamide (CAA) followed by heating at 95°C for 10 min. Prewashed magnetic PureCube Carboxy agarose beads (20 µL, Cube Biotech) were added to all the samples along with ACN (70% [v/v] final concentration) and incubated at ambient temperature for 20 min. Samples were placed on a magnetic rack and supernatants were discarded, and beads were washed twice with 70% (v/v) ethanol and once with neat ACN. ACN was completely evaporated from the tubes using a CentriVap (Labconco) before the addition of digestion buffer (50 mM Tris-HCl, pH 8) containing 1 µg each of enzymes Lys-C (Wako, 129–02541) and SOLu-Trypsin (Sigma-Aldrich, EMS0004). Trypsin-LysC on-bead digestion was performed with agitation (400 rpm) for 1 h at 37°C on a ThermoMixer C (Eppendorf). For global proteomics, the samples were transferred to pre-equilibrated C18 StageTips (2× plugs of 3M Empore resin, no. 2215) following digestion for sample clean-up. The eluates were lyophilised to dryness before being reconstituted in 150 µL 0.1% (v/v) FA / 2% (v/v) ACN ready for MS analysis. Peptides were loaded on a 15 cm C18 fused silica column with an integrated emitter tip (IonOpticks, ID 75 µm, OD 360 µm, 1.6 µm C18 beads) which was maintained at 50°C using a column oven. siRNA KD samples were acquired with a Neo Vanquish (Thermo Fisher Scientific) directly coupled online with an Astral mass spectrometer (Thermo Fisher Scientific) and peptides were separated with a binary buffer system of buffer A (0.1% [v/v] FA) and buffer B (80% [v/v] ACN plus 0.1% [v/v] FA), at a flow rate of 400 nL/min. The gradient started at 2% B and increased to 34% B in 30 min before increasing to 100% (v/v) B within 0.1 min and held for 3 min prior to returning to 2% (v/v) B and re-equilibration. The mass spectrometer was operated in positive polarity mode with a capillary temperature of 275°C. The DIA methods consisted of a MS1 scan (m/z = 380-980) with an AGC target of 5 × 106 and a maximum injection time of 5 ms (R = 240,000). DIA scans were acquired with the Astral detector with an AGC target of 8 × 104 and 3 ms maximum time. Fragmentation occurred in the higher-energy collisional dissociation (HCD) cell with a normalised stepped collision energy was 25% and the spectra were recorded in profile mode. 199 non-uniform DIA windows across 380-980 were collected with a maximum injection time of 3 ms and a 0.6 s loop control which achieved an average of five data points per peak. Global proteomic samples were acquired using a custom nano-flow high-performance liquid chromatography (HPLC) system (Thermo Fisher Scientific Ultimate 300 RSLC Nano-LC, PAL systems CTC autosampler). The HPLC was coupled to a timsTOF Pro (Bruker) equipped with a CaptiveSpray source. Peptides were loaded directly onto the column at a flow rate of 600 nL/min with buffer A (99.9% Milli-Q H2O, 0.1% [v/v] FA) and eluted at 400 nL/min on a 30 min linear analytical gradient of increasing buffer B (90% [v/v] ACN, 0.1% [v/v] FA) from 2% (v/v) to 34% (v/v). The timsTOF Pro MS was operated in diaPASEF mode using Compass Hystar 5.1. The settings on the thermal ionization MS (TIMS) analyser were as follows: Lock Duty Cycle to 100% with equal accumulation and ramp times of 100 ms, and 1/K0 Start 0.6 Vs/cm2 End 1.6 Vs/cm2, Capillary Voltage 1400 V, Dry Gas 3 L/min, Dry Temp 180°C. The DIA methods were set up using the instrument firmware (timsTOF control 2.0.18.0) for data-independent isolation of multiple precursor windows within a single TIMS scan. The method included two windows in each diaPASEF scan, with window placement overlapping the diagonal scan line for doubly and triply charged peptides in the m/z – ion mobility plane across 16 × 25 m/z precursor isolation windows (resulting in 32 windows) defined from m/z 400 to 1,200, with 1 Da overlap, and collision induced dissociation (CID) collision energy ramped stepwise from 20 eV at 0.8 Vs/cm2 to 59 eV at 1.3 Vs/cm2.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "", "otherOmicsLinks" : [ "pride.project:PXD073869", "pride.project:PXD074729", "pride.project:PXD062754", "pride.project:PXD062674", "pride.project:PXD073775", "pride.project:PXD074545", "pride.project:PXD073754", "pride.project:PXD073798", "pride.project:PXD074673" ], "submissionType" : "PARTIAL", "publicationDate" : "2026-04-09", "updatedDate" : "2026-03-05", "submissionDate" : "2026-03-05", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Philipp Schenk" ], "labPIs" : [ "David Komander" ], "affiliations" : [ "WEHI" ], "instruments" : [ "Orbitrap Astral" ], "softwares" : [ "DIA-NN" ], "quantificationMethods" : [ ], "sampleAttributes" : [ "cell culture", "Homo sapiens (Human)", "colon cancer", "mesenchymal cell", "colon endothelial cell", "breast cancer" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Cell culture", "Colon endothelial cell", "Mesenchymal cell" ], "diseases" : [ "Colon cancer", "Breast cancer" ], "references" : [ "Schenk P, Devine SM, Cobbold SA, Geoghegan ND, Kyran EL, Ang CS, Alexandrovics JA, Calleja DJ, Multari DH, Vaibhav V, Lu BGC, Klemm TA, Dagley LF, Lowes KN, Williamson NA, Eichhorn PJA, Ng AP, Feltham R, Komander D. Global analysis of cancer cell responses to USP9X inhibition. EMBO J. 2026--pubMed:41946992--doi: 10.1038/s44318-026-00742-y" ], "experimentTypes" : [ "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "P34.raw", "P18.raw", "P51.raw", "P25.raw", "P42.raw", "checksum.txt", "P35.raw", "P60.raw", "P43.raw", "P26.raw", "P52.raw", "P17.raw", "P28.raw", "report.pr_matrix.tsv", "P58.raw", "P31.raw", "P44.raw", "P57.raw", "P27.raw", "P14.raw", "P29.raw", "P16.raw", "P46.raw", "P59.raw", "P33.raw", "P20.raw", "P32.raw", "P50.raw", "P45.raw", "P15.raw", "P47.raw", "P39.raw", "P21.raw", "P38.raw", "P12.raw", "P55.raw", "P30.raw", "P48.raw", "P22.raw", "P56.raw", "P13.raw", "P53.raw", "P23.raw", "P36.raw", "P49.raw", "P40.raw", "P41.raw", "report.pg_matrix.tsv", "P11.raw", "P24.raw", "P37.raw", "P19.raw", "P54.raw" ], "highlights" : { } }, { "accession" : "PXD075265", "title" : "Uridine restores oocyte quality and mitigates female reproductive aging by inhibition of ferroptosis in mice", "projectDescription" : "Advanced maternal age is a key factor in female infertility, primarily due to declines in ovarian reserve and oocyte quality. However, the metabolic mechanisms underlying reproductive aging remain unclear. Here, we show that uridine levels in the plasma and ovaries of aged mice are significantly reduced compared with young controls. Building on this, we find that uridine supplementation significantly improves meiotic maturation, fertilization, and early embryonic development of aged oocytes, both in vivo and in vitro. Further microtranscriptomic analyses reveal that uridine enhances oocyte quality by inhibiting ferroptosis and enhancing mitochondrial function. Moreover, integrating Limited Proteolysis-Small Molecule Mapping, western blot and siRNA-based functional assays, we identify that uridine binds Poly(rC)-binding protein 1, thereby suppressing ferroptosis and preserving mitochondrial function. Collectively, these findings demonstrate that uridine supplementation improves fertility in aged female mice and provide mechanistic insight into ferroptosis in oocyte aging.", "dataProcessingProtocol" : "The MS/MS raw data were processed using SpectroMine software (v4.2.230428.52329) with the built-in Pulsar search engine against the UniProt FASTA database for peptide identification. Only peptides detected in more than 50% of biological replicates within at least one experimental group were retained for downstream analysis.", "sampleProcessingProtocol" : "Ovarian tissues collected from aged mice were homogenized and centrifuged at 11,270 × g for 10 min at 4°C, and the supernatant was collected. The resulting protein lysate originated from a single extraction and was equally divided into a control group and an incubation-treated group, which were subjected to independent incubation under their respective conditions. Protein concentrations were measured using a bicinchoninic acid (BCA) assay kit. The protein solution was adjusted to 600 μL and aliquoted into six equivalent volumes, each containing 100 μg of protein. For treatment, 0.33 nmol/μg (total protein) of uridine was added to three independent replicates of the treatment group and incubated for 10 min at 25°C, while dimethyl sulfoxide (DMSO) was added to the other three replicates as a control. Proteinase K was added simultaneously to all samples at a 1:100 enzyme/substrate ratio and incubated at 25°C for 5 min. Reactions were terminated by heating at 98°C for 2 min to inactivate Proteinase K, followed by reduction with 10 mM dithiothreitol at 37°C for 30 min and alkylation with 40 mM iodoacetamide at 37°C for 45 min in the dark. Proteins were further digested with trypsin at a 1:50 enzyme/substrate ratio overnight at 37°C under agitation at 78 × g to produce peptides for mass spectrometry analysis. The digestion was terminated by adding trifluoroacetic acid to adjust the pH to below 3. The resulting peptide mixtures were desalted using C18 spin columns, vacuum-dried, and reconstituted in 0.1% formic acid. The peptides were analyzed using a nano-UPLC system coupled to a timsTOF Pro2 mass spectrometer. The relevant services were provided by Shanghai Biotree Biotech.", "projectTags" : [ ], "keywords" : [ "Pcbp1", "Uridine", "Ferroptosis", "Oocyte quality", "Advanced maternal age" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-05", "updatedDate" : "2026-03-05", "submissionDate" : "2026-03-05", "downloadCount" : 41, "avgDownloadsPerFile" : 5.125, "botCount" : 14, "hubCount" : 26, "organicCount" : 1, "percentile" : 3, "yearlyDownloads" : [ { "year" : "2026", "count" : 41 } ], "submitters" : [ "JING CHEN" ], "labPIs" : [ "Jingyue Chen" ], "affiliations" : [ "Peking University Third Hospital No. 49 North Garden Road, Haidian District Beijing 100191, China" ], "instruments" : [ "6220 Time-of-Flight LC/MS" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "ovary", "Mus musculus (Mouse)" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Ovary" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "ovary_uridine_3.raw", "checksum.txt", "search.xlsx", "ovary_uridine_1.raw", "ovary_uridine_2.raw", "ovary_control_3.raw", "ovary_control_2.raw", "ovary_control_1.raw" ], "highlights" : { } }, { "accession" : "PXD075266", "title" : "ALR Couples IMS Redox and Heme Biosynthesis Beyond the Disulfide Relay", "projectDescription" : "Denaturing pulldown of ALR-HA from HEK293 cells to identify the covalent interaction partners of ALR. One half of the samples were treated with DTT during the Co-IP protocol to verify that the interactions are disulfide related. Accordingly, no Mock-cell lines was used. One half of the samples from the same cell line were treated with DTT during the IP and the other half was not.", "dataProcessingProtocol" : "Samples were analyzed by the CECAD Proteomics Facility on an Orbitrap Exploris 480 (Thermo Scientific, granted by the German Research Foundation under INST 216/1163-1 FUGG) mass spectrometer that was coupled to an Vanquish neo in trap-and-elute setup (Thermo Scientific). Samples were loaded onto a precolumn (Acclaim 5µm PepMap 300 µ Cartridge) with a flow of 60 µl/min before reverse-flushed onto an in-house packed analytical column (30 cm length, 75 µm inner diameter, filled with 2.7 µm Poroshell EC120 C18, Agilent). Peptides were chromatographically separated with an initial flow rate of 400 nL/min and the following gradient: initial 2% B (0.1% formic acid in 80 % acetonitrile), up to 6 % in 3 min. Then, flow was reduced to 300 nl/min and B increased to 20% B in 26 min, up to 35% B within 15 min and up to 98% solvent B within 1.0 min while again increasing the flow to 400 nl/min, followed by column wash with 95% solvent B and reequilibration to initial condition. The mass spectrometer was operated in data-dependent acquisition with a cycle time of 1 s with MS1 scans acquired from 350 m/z to 1400 m/z at 60k resolution and an AGC target of 300%. MS2 scans were acquired at 15 k resolution with a maximum injection time of 118 ms and an normalized AGC target of 50% in a 2 Th window and a fixed first mass of 110 m/z. All MS1 scans were stored as profile, all MS2 scans as centroid. All mass spectrometric raw data were processed with Maxquant (version 2.6, Tyanova et al. NatProt) using default parameters against the Uniprot canonical human database (UP5640, downloaded 15.01.2025) with the match-between-runs option enabled between replicates. Follow-up analysis was done in Perseus 1.6.15 (Tyanova et al. NatMet). Protein groups were filtered for potential contaminants and insecure identifications. Remaining IDs were filtered for data completeness in at least one group and missing values imputed by sigma downshift (0.3 σ width, 1.8 σ downshift). Afterwards, FDR-controlled two-sided t-tests were performed.", "sampleProcessingProtocol" : "For the interactome data an in-gel digest was performed. The native IP was performed as described in the respective section. After the native IP was performed the beads were dried and boiled in 20 µl reducing Laemmli buffer (without bromphenolblue) for 10 min. The samples were reduced by addition of DTT with a final concentration of 5 mM and incubated at 56 °C for 30 min. Free cysteine thiols were alkylated by addition of CAA to a final concentration of 40 mM to the samples which were then incubated for 30 min at room temperature in the dark. The samples were run on SDS-PAGE until the samples migrated for 1 cm into the separation gel. Then the gels were fixed for 1 h in fixing solution (10% Acetic acid / 20% Methanol in water). The gel bands were cut in smaller pieces and were transferred to individual tubes. 100 µl of 50 mM ABC/50% Acetonitrile was added to the gel pieces and were incubated for 20 min at room temperature. The solution was exchanged with fresh 50 mM ABC/50% Acetonitrile and remaining solution was discarded after 20 min incubation. The gel pieces were covered with 100 µl acetonitrile and incubated for 10 min. The gel pieces were then dried in a speedvac for approximately 5 min. A digestion solution of 10 ng/μL of 90 % trypsin and 10 % LysC in 50 mM ammonium bicarbonate (ABC) was added to the gel pieces until the gel pieces were fully covered. The gel pieces were incubated for 30 min at 4°C with the digest solution. After the incubation time, excessive digest solution was removed and 50 mM ABC buffer was used to cover the gel pieces. The samples were incubated overnight at 37°C while shaking at 750 rpm. The next day, the supernatant of the gel pieces were transferred into new tubes. The gel pieces were covered with 100 µl 30% ACN/3% TFA and incubated for 20 min at room temperature. The extract was combined with the supernatant of the previous step. The gel pieces were covered with 100 µl 100% acetonitrile and again incubated for 20 min at room temperature. The extract was also combined with the supernatant from the previous step and the organic solvents of the samples were reduced in the speedvac until a remaining volume of 50 µl was reached. The samples were acidified by addition of formic acid to a final concentration of 1% and the STAGE tip purification protocol as it is described in the section “Determination of cellular protein levels by quantitative label-free proteomics” was performed. The STAGE tips were stored at 4°C. The mass spectrometry was performed and analysed by the proteomics core facility Cologne.", "projectTags" : [ ], "keywords" : [ "Mitochondria", "Oxidative protein folding", "Heme biosynthesis" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-16", "updatedDate" : "2026-03-05", "submissionDate" : "2026-03-05", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Proteomics Facility" ], "labPIs" : [ "Jan Riemer" ], "affiliations" : [ "Redox Metabolism, Institute for Biochemistry, University of Cologne" ], "instruments" : [ "Orbitrap Exploris 480" ], "softwares" : [ ], "quantificationMethods" : [ "Label free" ], "sampleAttributes" : [ "cell culture", "Homo sapiens (Human)" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Cell culture" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "E3_4586_03.raw", "PRIDE_4554.zip", "E2_4554_02.raw", "E3_4586_08.raw", "E3_4586_05.raw", "PRIDE_4586.zip", "E2_4554_04.raw", "E2_4554_06.raw", "E2_4554_08.raw", "checksum.txt", "E2_4554_03.raw", "E3_4586_07.raw", "E3_4586_02.raw", "E2_4554_01.raw", "E3_4586_04.raw", "E3_4586_06.raw", "E2_4554_05.raw", "E3_4586_01.raw", "E2_4554_07.raw" ], "highlights" : { } }, { "accession" : "PXD075247", "title" : "Competitive kinobead profiling of RAF inhibitors", "projectDescription" : "Kinase enrichment was performed using kinobeads followed by LC–MS/MS to profile target engagement/competition by RAF inhibitors. HEK293T lysates overexpressing 185 protein kinases were prepared in modified RIPA buffer, pre-incubated with DMSO or inhibitor, then incubated with kinobeads to enrich kinases. Beads were washed, proteins denatured, and on-bead digestion was performed (Lys-C/Trypsin). Resulting peptides were cleaned up and analyzed by LC–MS/MS.", "dataProcessingProtocol" : "Raw datasets were analyzed using the FragPipe suite with DIA-NN enabled (variable mods Oxidation (M) and Acetyl (protein N-term); FDR 0.01; searched against Human proteome FASTA downloaded from UniProt). Protein abundances from the DIA-NN gene groups output were used for downstream processing. Raw intensities were transformed to calculate enrichment levels relative to DMSO by using log2[Intensity(DMSO)/Intensity(inhibitor)] and significance was assessed by two-sample t-tests (FDR 0.05).", "sampleProcessingProtocol" : "Biological system: HEK293T-derived line overexpressing 185 protein kinases Lysis buffer: modified RIPA (50 mM Tris pH 7.8, 150 mM NaCl, 10 mM NaF, 1% Igepal CA-630, 0.25% sodium deoxycholate, 5% glycerol) + protease inhibitor, 1 mM PMSF, phosphatase inhibitor cocktail 2&3. Lysate clarification: 17,000 × g, 10 min, 4 °C. Protein conc.: quantified by Pierce 660 nm assay; diluted to 2.0 mg/mL. Competition step: lysate (160 µL) incubated with DMSO or 10 µM inhibitor, 30 min, 4 °C, end-over-end rotation. Kinobead enrichment: 10 µL of 50% kinobead slurry washed twice; inhibitor-treated lysate incubated with kinobeads 3 h, 4 °C, end-over-end rotation. Washing: Post-enrichment, beads washed twice with RIPA and three times with 1X TBS Digestion: Remaining bead-bound proteins denatured and digested with Lys-C / Trypsin MS processing: Library-free DIA", "projectTags" : [ ], "keywords" : [ "Human", "Lc-ms", "Kinase" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-20", "updatedDate" : "2026-03-05", "submissionDate" : "2026-03-05", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Daniel Brush" ], "labPIs" : [ "Dustin James Maly" ], "affiliations" : [ "Department of Chemistry, University of Washington" ], "instruments" : [ "Orbitrap Fusion Lumos" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "kidney", "Homo sapiens (Human)" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Kidney" ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.1038/S41589-026-02212-2" ], "experimentTypes" : [ "Data-independent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "report.pr_matrix.tsv", "A20250523_DB_DIA_DMSO_a.raw", "A20250523_DB_DIA_LXH_b.raw", "A20250523_DB_DIA_TOVO_b.raw", "A20250523_DB_DIA_AZ_c.raw", "A20250523_DB_DIA_AZ_a.raw", "A20250523_DB_DIA_LXH_a.raw", "A20250523_DB_DIA_LF_b.raw", "report.tsv", "A20250523_DB_DIA_LXH_c.raw", "checksum.txt", "report.pg_matrix.tsv", "A20250523_DB_DIA_LF_c.raw", "A20250523_DB_DIA_TOVO_a.raw", "UNIPROT_HUMAN_DIA.fasta", "A20250523_DB_DIA_TOVO_c.raw", "A20250523_DB_DIA_DMSO_b.raw", "A20250523_DB_DIA_DMSO_c.raw", "A20250523_DB_DIA_LF_a.raw", "A20250523_DB_DIA_AZ_b.raw" ], "highlights" : { } }, { "accession" : "PXD075198", "title" : "Lyophilization prior to homogenisation and extraction increases protein yield and membrane protein detection in Gram-negative bacterial proteomic analyses", "projectDescription" : "Multi-drug resistant Gram-negative bacteria (GNB) are major contributors to the anti-microbial resistance (AMR) burden in humans and animals. AMR mechanisms are primarily mediated by proteoforms and, therefore, proteomic analyses of GNB offers a significant advantage in understanding the mechanisms of AMR. A large portion of these mechanisms are mediated by membrane proteins, however, they are often difficult to extract due to their hydrophobic nature and complex interactions with other components of the cell membrane. To extract the greatest number of proteoforms, an efficient homogenisation protocol is required to effectively disrupt the rigid cell wall and membrane. Bead-beating has previously been demonstrated to increase cellular disruption and improve protein extraction yields in mammalian systems, however, the efficiency of bead-beating is dependent on various factors, including cell structure characteristics. Using Escherichia coli and Klebsiella pneumoniae, we systematically compared the extraction efficiency of bead-beating with flash frozen and lyophilized cell pellets. We demonstrate that lyophilization prior to homogenisation by bead-beating increases protein extraction yield, and increases the detection of hydrophobic, membrane proteins. We detected numerous unique membrane proteins in each bacterial isolate, including ABC transporters, multi-drug efflux pumps, and proteins involved in lipopolysaccharide (LPS) synthesis, when lyophilizing prior to bead-beating compared to only flash freezing prior to bead-beating. As membrane proteins play a central role in AMR resistance mechanisms, this improvement in their isolation and identification will aid in under-standing the resistance and molecular mechanisms associated with multi-drug resistant GNB.", "dataProcessingProtocol" : "The MS raw files were searched using Peaks Studio 13 (Bioinformatic Solutions Inc.) against the Escherichia coli K12 UniProt database (UP000000625, date accessed 10 October 2025) or the Klebsiella pneumoniae HS11286 UniProt database (UP000007841, date accessed 11 August 2025), and a database of common contaminants with the following parameters: Precursor mass error tolerance: 10.00 ppm. Fragment mass error tolerance: 0.02 Da. Enzyme: Trypsin. Maximum missed cleavages: 4. Digest-mode: Semi-specific. Peptide length range: 6–45. Fixed modifications: none. Variable modifications: Propio-namide (Cysteine), Oxidation (Methionine), Deamidation (Asparagine and Glutamine), Carbamylation (N-terminal Lysine). Maximum variable PTM per peptide: 4. Peptide spectrum match (PSM) false discovery rate (FDR): 1.0%. Protein Group FDR: 1.0%. Unique peptides: ≥ 1.", "sampleProcessingProtocol" : "The E. coli strain used in this study is strain MG1655, the laboratory strain of E. coli K-12, hence referred to as Coli K12. The K. pneumoniae isolates used in this study, hence referred to as KC32 and KC89, are two clinical isolates obtained from patients with urinary tract infections treated at Concord Repatriation General Hospital in Sydney, Australia. Bacterial isolates were cultured in Lysogeny Broth (LB) broth at 37 °C and 200 rpm in five biological replicates (with the exception of KC89 where n=3) and grown to late exponential/early stationary phase (OD600 ≈ 1.30). Each culture was divided into two 20 mL aliquots, followed by harvesting by centrifugation at 10000 g for 5 minutes. The supernatant of each aliquot was discarded. The cell pellet of one aliquot from each biological replicate was lyophilized using a Christ Alpha 2-4 LDplus Laboratory Freeze Dryer, and the cell pellet of the second aliquot from each biological replicate was flash frozen with liquid nitrogen. The lyophilized and flash frozen cell pellets were disrupted and pulverized using a Benchmark Scientific BeadBug Benchtop Microtube Homogeniser for 3 x 30 seconds at 4000 rpm, with the sample placed on ice for 30 seconds between rounds. The resulting powders were suspended in UTC7 extraction buffer (7 M Urea, 2 M Thiourea, 1% C7BzO, 100 mM Tris-HCl) supplemented with 1X Roche cOmplete™ Mini EDTA-free Protease Inhibitor (PI) Cocktail and 50 mM LiCl, then sonicated in a bath sonicator for 10 minutes. Both extracts were treated with 1 μL benzonase, bath sonicated for a further 10 minutes, followed by a 50 minute incubation period at room temperature to shear DNA, thereby reducing viscosity of the extracts. The extracts were reduced with 100 mM DTT + 5 mM TBP for 1 h, followed by alkylation with 220 mM of acrylamide for 1 h, at room temperature. Insoluble cellular debris was removed by centrifugation for 10 minutes at 16873 g. Protein quantification was performed using a paper-based assay. Fifty micrograms of protein was diluted 10-fold with 100 mM Tris-HCl to reduce the concentration of urea to >1 M and digested with 500 ng sequencing grade trypsin (100:1 protein:enzyme ratio) at 37 °C for 16 hours. Peptides were recovered using SDB-RPS-based STAGE tip. Eluted peptides were evaporated to dryness using a SavantTM DNA 120 SpeedVac Concentrator (Thermo Fisher Scientific) and reconstituted in 100 µL of MS loading solvent (2% (v/v) acetonitrile, 0.2% (v/v) trifluoroacetic acid) for a final concentration of 100 ng/µL. Using a Waters Acquity M-class nanoLC system, 500 ng of the sample in 5 µL was loaded at 15 μL/min for 2 min onto a nanoEase Symmetry C18 trapping column (180 μm × 20 mm; Waters) before being washed onto an Aurora Elite TS C18 column (150 mm x 75 μm ID, 1.6 μm C18 particle size; Ion Opticks, Fitzroy, Australia) heated to 45°C. Peptides were eluted from the column and into the source of a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) using the following gradient: 5%–30% MS buffer B (100% ac-etonitrile) and 95%-70% MS buffer A (0.2% Formic acid in water) over 120 min, 30%–80% MS buffer B over 2 min, 80% MS buffer B for 2 min, and 80%–5% over 2 min. The eluting peptides were ionized at 2100 V. A Data Dependent MS/MS (dd-MS2) experiment was performed, with a survey scan of 350–1,500 m/z performed at 70,000 resolution for pep-tides of charge state 2+ or higher with an AGC target of 3e6 and maximum injection time of 50 ms. The top 12 peptides were selected and fragmented in the HCD cell using an isolation window of 1.4 m/z, an AGC target of 1e5 and maximum injection time of 100 ms. Fragments were scanned in the Orbitrap analyser at 17,500 resolution and the product ion fragment masses measured over a mass range of 120–2000 m/z. The mass of the precursor peptide was then excluded for 5 s.", "projectTags" : [ ], "keywords" : [ "Proteomics", "Antimicrobial resistance", "Bacterial protein extraction", "Membrane proteins" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-08", "updatedDate" : "2026-03-04", "submissionDate" : "2026-03-04", "downloadCount" : 129, "avgDownloadsPerFile" : 3.7941177, "botCount" : 9, "hubCount" : 119, "organicCount" : 1, "percentile" : 6, "yearlyDownloads" : [ { "year" : "2026", "count" : 129 } ], "submitters" : [ "Breyer-Lynne Woodland" ], "labPIs" : [ "Matthew P. Padula" ], "affiliations" : [ "School of Life Sciences and Proteomics and Metabolomics Core Facility, Faculty of Science, University of Technology Sydney, Ultimo, NSW 2007, Australia" ], "instruments" : [ "Q Exactive Plus" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Escherichia coli" ], "organisms" : [ "Escherichia coli" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "SampleID_MSFileName_Legend.csv", "KC32_PeaksStudio_db.peptides.csv", "20250929_KC32_2B.raw", "20250929_ColiK12_1A.raw", "20250929_KC32_5B.raw", "20250929_ColiK12_1B.raw", "20250929_KC32_3A.raw", "20250929_ColiK12_4B.raw", "20250722_KC89_1A.raw", "checksum.txt", "20250722_KC89_3B.raw", "20250929_KC32_3B.raw", "20250929_ColiK12_3B.raw", "20250929_ColiK12_4A.raw", "20250722_KC89_2B.raw", "20250929_ColiK12_5B.raw", "20250929_KC32_4A.raw", "20250929_KC32_1A.raw", "20250929_ColiK12_2B.raw", "20250722_KC89_3A.raw", "20250929_ColiK12_3A.raw", "ColiK12_PeaksStudio_db.proteins.csv", "KC32_PeaksStudio_db.proteins.csv", "20250929_KC32_4B.raw", "20250722_KC89_2A.raw", "20250929_KC32_1B.raw", "20250929_ColiK12_5A.raw", "KC89_PeaksStudio_db.peptides.csv", "20250929_ColiK12_2A.raw", "20250929_KC32_5A.raw", "KC89_PeaksStudio_db.proteins.csv", "20250929_KC32_2A.raw", "ColiK12_PeaksStudio_db.peptides.csv", "20250722_KC89_1B.raw" ], "highlights" : { } }, { "accession" : "PXD075197", "title" : "Salmonella-derived heme inhibits macrophage phagocytosis and promotes infection in mice", "projectDescription" : "Bacterial pathogens such as Salmonella enterica serovar Typhimurium can resist phagocytosis by macrophages. Here we explored the role of bacterial heme biosynthesis in phagocytosis resistance. Using transposon sequencing (Tn-seq) during Salmonella infection of macrophages, we identify a methyltransferase, SirM, that indirectly inhibits phagocytosis of bacteria. Mechanistically, sirM is activated upon interaction with macrophages and methylates HemL, a key enzyme in heme biosynthesis, resulting in upregulation of heme synthesis by Salmonella. Salmonella-derived heme inhibits Cdc42 activation in a Toll-like receptor 4 (TLR4)-dependent manner to inhibit phagocytosis. Moreover, sirM promotes macrophage death by increasing heme synthesis. Experiments in mouse models show that sirM is required for virulence and confers a competitive advantage over intestinal commensal bacteria during infection. We also found that sirM is distributed among enteric pathogens. Collectively, our findings show that bacterial heme promotes evasion of phagocyte responses and pathogenesis to confer an advantage in the host.", "dataProcessingProtocol" : "All MS/MS spectra were searched against the UniProt Salmonella Proteome database (Salmonella Typhimurium 14028s, Proteome ID: UP000002695) using PEAKS 11.0 (Bioinformatics Solutions) for de novo sequencing and database searching. The precursor and fragment mass error tolerances were set to 10 ppm and 0.02 Da, respectively. Semi-trypsin was specified as the digestion enzyme, allowing up to two missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification, while oxidation of methionine and acetylation of the N-terminus were set as variable modifications. Peptide-spectrum matches (PSMs) were filtered with a −10logP score ≥ 20, and proteins were filtered with a −10logP score ≥ 15 and at least one unique peptide, resulting in an estimated FDR of less than 1% at the PSM level. Proteins sharing significant peptide evidence were grouped into clusters.", "sampleProcessingProtocol" : "The IP sample was denatured in 2% SDS buffer containing 50 mM DTT for 20 min at room temperature (RT), followed by boiling at 100°C for 5 min. Proteins were alkylated in the dark for 1 h at RT with 200 mM iodoacetamide (IAA). Subsequently, five volumes of pre-cooled acetone were added, and proteins were precipitated overnight at -20°C. The protein pellet was resolubilized and digested with sequencing-grade modified trypsin (Promega) at an enzyme-to-protein ratio of 1:50 (w/w) overnight at 37°C. Tryptic peptides were collected by centrifugation at 14,000 g for 20 min at 20°C, acidified with 1% trifluoroacetic acid (TFA), and desalted using C18 Ziptips. Peptides were eluted with 0.1% TFA in 50-70% acetonitrile, lyophilized in a SpeedVac (ThermoSavant), and resuspended in 10 µL of 1% formic acid/5% acetonitrile. All mass spectrometric experiments were performed on a Thermo Fusion Lumos mass spectrometer coupled to an Easy-nLC 1200 system via an Easy Spray source (Thermo Fisher Scientific). Peptide mixtures were loaded onto a 15 cm × 75 µm i.d. column packed with C18 2-µm reversed-phase resins (PepMap RSLC) and separated over a 60 min linear gradient from 95% solvent A (0.1% formic acid/2% acetonitrile) to 28% solvent B (0.1% formic acid/80% acetonitrile) at a flow rate of 300 nL/min. The spray voltage was set to 2.1 kV, ion transfer capillary temperature to 275°C, and RF lens to 60%. The mass spectrometer was operated in positive ion mode and data-dependent acquisition (DDA) mode using Tune and Xcalibur 4.0.27.19. Full MS scans (m/z 350-1500) were acquired at a resolution of 60,000 (at m/z 400), followed by fragmentation of the twenty most abundant multiply charged ions (charge states 2-7; excluding singly charged and unassigned ions) with collision energy of 30%. Dynamic exclusion was enabled with default settings.", "projectTags" : [ ], "keywords" : [ "Macrophage; phagocytosis; methyltransferase; heme; tlr4" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-07", "updatedDate" : "2026-03-04", "submissionDate" : "2026-03-04", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Zuoqiang Wang" ], "labPIs" : [ "Yu-Feng Yao" ], "affiliations" : [ "Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine." ], "instruments" : [ "Orbitrap Fusion Lumos" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)" ], "organisms" : [ "Salmonella typhimurium (strain lt2 / sgsc1412 / atcc 700720)" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "SirM.raw", "proteins.csv", "CK.raw" ], "highlights" : { } }, { "accession" : "PXD075222", "title" : "Pull-down experiment of Cdkn1a mRNA and its protein binding partners in wounds and homeostatic skin of porcine skin", "projectDescription" : "To elucidate the mechanism responsible for the absence of p21 (protein) in homeostatic porcine skin and its rapid generation upon injury, we performed a pull-down experiment of Cdkn1a mRNA and its protein binding partners. For this, we utilized a variant of the comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS), which has been successfully used in human cells for large noncoding RNAs, but never before applied to tissues in vivo or to small targets such as Cdkn1a mRNA", "dataProcessingProtocol" : "The RAW MS data were analyzed with FragPipe (22.0), using MSFragger (4.1), IonQuant (1.10.27, and Philosopher (5.0.0). The default FragPipe workflow for label free quantification (LFQ-MBR) was used, except “Normalize intensity across runs” was turned off. Cleavage specificity was set to Trypsin/P, with two missed cleavages allowed. The protein FDR was set to 1%. A mass of 57.02146 (carbamidomethyl) was used as fixed cysteine modification; methionine oxidation and protein N-terminal acetylation were specified as variable modifications. MS2 spectra were searched against the Sus scrofa 1 protein per gene reference proteome from Uniprot (Proteome ID: UP000008227, release 2024_04), concatenated with a database of 382 common laboratory contaminants (release 2023.03, https://github.com/maxperutzlabs-ms/perutz-ms-contaminants) and two additional protein sequences corresponding to the expressed transgenic constructs. Computational analysis was performed using Python and the in-house developed Python library MsReport (version 0.0.26). Only non-contaminant proteins identified with a minimum of two peptides and being quantified in at least one replicate of one experiment were considered for the analysis. LFQ protein intensities reported by FragPipe were log2-transformed and normalized across samples using the ModeNormalizer from MsReport. This method involves calculating log2 protein ratios for all pairs of samples and determining normalization factors based on the modes of all ratio distributions. Missing values were imputed with fixed values close to the detection limit. The in-house Python library XlsxReport (0.0.8) was used to create a formatted Excel file summarizing the results of protein quantification.", "sampleProcessingProtocol" : "Three different types of pig skin samples were collected: control skin, biopsy punches from the proximity of excision wounds, and microneedled skin areas. For generation of wounded skin samples, pig skin undergone either excision injury with a 4 mm biopsy punch or microneedled 50 times at a depth of 3 mm using a DermaStamp. Excision wound samples were generated by collecting a 6 mm biopsy punch from around the excision wound, resulting in donut (ring)-shaped samples representing ~1 mm of tissue around the injury site. All wounded skin samples were collected 1.5 hours post-injury. Control and microneedled skin samples were collected using a 4 mm biopsy punch. All the biopsies were removed using tweezers and sharp scissors. The biopsy was washed in PBS, and the dermal layer was trimmed away as much as possible with a razor blade. Each biopsy punch was then immediately fixed in 4% formaldehyde for 6 hours. After the fixation, the samples were washed three times with 50 mL of chilled PBS. Fifty biopsy samples per group were ground in liquid nitrogen with a mortar and pestle, then transferred to 50 mL Falcon tubes containing 10× volume lysis buffer (50 mM Tris-Cl pH 7.0, 10 mM EDTA, 1% SDS, PMSF, protease inhibitors, and Superase-In). Samples were kept on ice and homogenized (ULTRA TURRAX T25) six times for 1 min at maximum speed, followed by sonication (Bandelin Sonoplus mini20) 20× for 1 min at 40% energy with 1 min rest intervals. Homogenates were incubated on ice for 45 min, centrifuged (16,100 RCF, 10 min, 4°C), and supernatants collected. Each was pre-cleared with Dynabeads MyOne Streptavidin C1 (30 µL beads/mL), incubated at 37°C, and washed twice. Hybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris-Cl pH 7.0, 1 mM EDTA, inhibitors) and 260 pmol raPOOL probe were added per sample and incubated overnight at 37°C. Fresh C1 beads (100 µL per 100 pmol probe) were added for 30 min binding at 37°C, washed five times with preheated SSC/SDS buffer, and eluted in biotin/HEPES buffer. After 20 min shaking at room temperature and 10 min at 65°C, beads were removed, and eluates were de-crosslinked overnight at 65°C. The 400µL samples were reduced and alkylated by adding 10 µL of 5 mM TCEP and 9.6µL 12mM iodoacetamide and incubating for 20 min at 25 ˚C at 1200rpm shaking in the dark. Sera-MagSpeed Beads mix (GE Life Sciences, cat. nos. 45152105050350 and 65152105050350) was added to the sample at beads:protein ratio of 10:1 and mixed with ethanol to real 50% final ethanol concentration. The tubes were kept in a thermomixer for 10min at 1000rpm at 23˚C to let protein bind to the beads. Then the supernatant was removed using a magnetic rack and the beads were washed with 500µL 80% ethanol in water off the rack. And the wash procedure was repeated 3 more times. Afterwards, 20µL 50mM ammonium bicarbonate with 200ng trypsin (Trypsin Gold, Promega) and 50ng LysC (mass spectrometry grade, FUJIFILM Wako chemicals) was added to the beads and incubated at 37°C 1000rpm overnight. The beads were settled on a magnetic rack and the supernatant was transferred to a new tube. The beads were rinsed with 8.5 µL ammonium bicarbonate and sonicated for 30s and the supernatant was pooled to the supernatant from the previous step. Then the samples were centrifugated at 20000g for 2min and the supernatant was transferred to a new tube to remove any remaining beads. Trifluoroacetic acid was added to reach a final concentration of 0.5 %. Peptides were separated on a Vanquish Neo nano-flow chromatography system (Thermo-Fisher), using a trap-elute method for sample loading (Acclaim PepMap C18, 2 cm × 0.1 mm, 5 μm, Thermo-Fisher), and a C18 analytical column (Acclaim PepMap C18, 50 cm × 0.75 mm, 2 μm, Thermo-Fisher), applying a segmented linear gradient from 2% to 35% and finally 80% solvent B (80 % acetonitrile, 0.1 % formic acid; solvent A 0.1 % formic acid) at a flow rate of 230 nL/min over 120min. Eluting peptides were analyzed on an Exploris 480 Orbitrap mass spectrometer (Thermo-Fisher Scientific) coupled to the column with a FAIMS pro ion-source (Thermo-Fisher Scientific) using coated emitter tips (PepSep, MSWil) with the following settings: The mass spectrometer was operated in DDA mode with two FAIMS compensation voltages (CV) set to -45 or -60 and 1.5 s cycle time per CV. The survey scans were obtained in a mass range of 350-1500 m/z, at a resolution of 60k at 200 m/z, and a normalized AGC target at 100%. The most intense ions were selected with an isolation width of 1.4 m/z, fragmented in the HCD cell at 30% collision energy, and the spectra recorded for max. 100 ms at a normalized AGC target of 100% and a resolution of 15k. Peptides with a charge of +2 to +6 were included for fragmentation, the peptide match feature was set to preferred, the exclude isotope feature was enabled, and selected precursors were dynamically excluded from repeated sampling for 45 seconds.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-31", "updatedDate" : "2026-03-04", "submissionDate" : "2026-03-04", "downloadCount" : 6, "avgDownloadsPerFile" : 0.5, "botCount" : 0, "hubCount" : 6, "organicCount" : 0, "percentile" : 1, "yearlyDownloads" : [ { "year" : "2026", "count" : 6 } ], "submitters" : [ "Markus Hartl" ], "labPIs" : [ "Mikolaj Ogrodnik" ], "affiliations" : [ "Ludwig Boltzmann Institute for Traumatology, The Research Center in Cooperation with AUVA, Austrian Cluster for Tissue Regeneration, The laboratory for Tissue Damage Responses in Regeneration and Aging,Donaueschingenstrasse 13, A-1200 Vienna, Austria," ], "instruments" : [ "Orbitrap Exploris 480" ], "softwares" : [ "FragPipe" ], "quantificationMethods" : [ "label-free quantification" ], "sampleAttributes" : [ "skin", "Sus scrofa domesticus (domestic pig)" ], "organisms" : [ "Sus scrofa domesticus (domestic pig)" ], "organismsPart" : [ "Skin" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "checksum.txt", "MsReport_Result.zip", "20240918_E2_NEO5_Rozmaric_Ogrodnik_MPLGmbH_Academic_MPLID24223_C4_DONUT_p21_50p.raw", "ID24223_Raw_file_experiment_list.xlsx", "20240918_E2_NEO5_Rozmaric_Ogrodnik_MPLGmbH_Academic_MPLID24223_C1_CTRL_LacZ_50p.raw", "FragPipe_Result.zip", "20240918_E2_NEO5_Rozmaric_Ogrodnik_MPLGmbH_Academic_MPLID24223_C3_DONUT_LacZ_50p.raw", "20240918_E2_NEO5_Rozmaric_Ogrodnik_MPLGmbH_Academic_MPLID24223_C2_CTRL_p21_50p.raw", "20240918_E2_NEO5_Rozmaric_Ogrodnik_MPLGmbH_Academic_MPLID24223_C5_MICRON_LacZ_50p.raw", "contaminants_2023_03.fasta", "20240918_E2_NEO5_Rozmaric_Ogrodnik_MPLGmbH_Academic_MPLID24223_C6_MICRON_p21_50p.raw", "2024_04_UP000008227_S_scrofa_Duroc_1ppg.fasta" ], "highlights" : { } }, { "accession" : "PXD075230", "title" : "Bulk proteomics of porcine skin excision injury at 15 minutes and 4 hours post wounding.", "projectDescription" : "We aimed to define the early molecular events of porcine skin excision injury by investigating at 15 minutes and 4 hours post-wounding. Our objective was to compare time points to identify proteins and pathways that initiate, sustain, or modulate the injury response, including signaling, cytoskeletal dynamics, inflammation, and early repair processes. Using TMT-based quantitative LC–MS/MS on porcine skin tissue, we sought to generate a high-confidence, time-resolved proteomic map that highlights key regulators and potential therapeutic targets in the immediate and short-term phases after injury. We preformed phospho-proteomics analysis to capture rapid, site-specific phosphorylation changes that report on signaling pathway activation.", "dataProcessingProtocol" : "Peptides were separated on an Ultimate 3000 RSLC nano-flow chromatography system (Thermo-Fisher), using a pre-column for sample loading (Acclaim PepMap C18, 5 mm × 300 µm, 5 μm, Thermo-Fisher) and a C18 analytical column (Acclaim PepMap C18, 50 cm × 75 µm, 2 μm, Thermo-Fisher), applying a segmented linear gradient from 2% to 35% and finally 80% solvent B (80 % acetonitrile, 0.1 % formic acid; solvent A 0.1 % formic acid) at a flow rate of 230 nL/min over 180 min. Eluting peptides were analyzed on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher) coupled to the column with a FAIMS pro ion-source (Thermo-Fisher) using coated emitter tips (PepSep, MSWil) with the following settings: The mass spectrometer was operated in DDA mode with three FAIMS compensation voltages (CV) set to -40, -55 or -70 and 2 s cycle time per CV. The survey scans were obtained in a mass range of 350-1500 m/z, at a resolution of 60k at 200 m/z, and a normalized AGC target at 100%. The most intense ions were selected with an isolation width of 0.7 m/z, fragmented in the HCD cell at 34% collision energy, and the spectra recorded for max. 100 ms at a normalized AGC target of 200%. Peptides with a charge of +2 to +6 were included for fragmentation, the peptide match feature was set to preferred, the exclude isotope feature was enabled, and selected precursors were dynamically excluded from repeated sampling for 45 seconds. Raw data were split into single cv using FreeStyle 1.8 SP2 and processed using the MaxQuant software package (version 2.1.4.0,) and the Uniprot Sus scrofa(pig) reference proteome (version 2022_04, www.uniprot.org), as well as a database of most common contaminants. The search was performed in MS2 TMT11 reporter ion mode with isobaric label correction, with full trypsin specificity and a maximum of two missed cleavages at a protein and peptide spectrum match false discovery rate of 1%. Carbamidomethylation of cysteine residues were set as fixed, oxidation of methionine and N-terminal acetylation as variable modifications. For phospho-enriched samples, additional variable modification phosphorylation(STY) was included. For label-free quantification the “match between runs” feature and the LFQ function were activated - all other parameters were left at default. MaxQuant output files were analyzed in R (4.2.2) using custom scripts. The analysis procedure covered: correction for isotopic impurities of labels, normalization, and statistical between-group comparisons using LIMMA. To account for differences of protein abundance on phosphorylation-site level, site intensities were normalized to protein intensities before group comparisons with LIMMA.", "sampleProcessingProtocol" : "For proteomic analysis of porcine excision-injury samples, tissue was collected from 3-4 pigs. In each animal, circular 6 mm full-thickness excision wounds were created, and the surrounding tissue was sampled using 8 mm punch biopsies at three time points: immediately after wounding (control; n=4), 15 minutes (“short”; n=4), and 4 hours (“long”; n=3). For each condition in each pig, two 8 mm biopsies were taken adjacent to the wound margin. Biopsies were briefly rinsed in ice-cold PBS and snap-frozen in liquid nitrogen immediately after collection, then stored at −80°C until lysis. The biopsies were resuspended in 500µL lysis buffer (6M urea, 50mM Tris-HCl pH 8.0, 150mM NaCl, 1mM PMSF; phosphatase inhibitor cocktail 2, phosphatase inhibitor cocktail 3), with sonication and vortex. Then 1µL benzonase was added and the samples were kept on ice for 5-10minutes. Then 20 pulses of sonication was applied, followed by another 15minutes on ice for further benzonase treatment. The lysates were centrifugated at 15,000 x g for 10mins at 10°C and the supernatants were transferred to new tubes. BCA assays was used for the protein concentration measurement. About 200µL samples were precipitate using 4x volumes of cold acetone. About 100µg protein pellets were resuspended in 30 µL 8 M urea and 50 mM ammonium bicarbonate. Disulfide bonds were reduced with 1.2 µL of 250 mM dithiothreitol (DTT) for 30 min at room temperature before adding 1.2 µL of 500 mM iodoacetamide and incubating for 30 min at room temperature in the dark. The remaining iodoacetamide was quenched with 0.6 µL of 250 mM DTT for 10 min. 30 µL 50 mM ammonium bicarbonate was added to the tube before proteins were digested with 1 µg LysC (Promega) at room temperature for 90 minutes. Then 180 µL 50 mM ammonium bicarbonate was added to dilute urea to 1M, before 2 µg trypsin (Trypsin Gold, Promega) was added and samples were kept at 37°C overnight. The digest was stopped by the addition of trifluoroacetic acid (TFA) to a final concentration of 0.5 %, and the peptides were desalted with Sep-Pak tC-18 cartridges (Waters). Desalted samples were dried for 30 min in a SpeedVac concentrator and subsequently lyophilized overnight. Lyophilized peptides were dissolved in 50 μl 100 mM TEAB (Sigma). 450 μg of each TMT-10Plex reagent (Thermo Fisher Scientific) were dissolved in 30 μl of 100% anhydrous acetonitrile and 30 μl was added to the peptide/TEAB mixes at 1:4 sample:label ratio. Labels used: 126C: pig33_15min; 127N: pig33_control; 127C: pig34_15min; 128N: pig34_control; 128C: pig35_15min; 129N: pig35_control; 129C: pig36_15min; 130N: pig36_control; 130C: pig33_long; 131N: pig34_long; 131C: pig36_long. Samples were labeled for 60 min at RT. 0.1 μl of each sample were pooled, mixed with 10 μl 0.1% TFA, and analyzed by mass spectrometry (MS) to confirm the labeling efficiency > 99%. For quenching, 8 μl of 5% hydroxylamine was added and the reaction was incubated for 25 min at RT. Samples were pooled and subsequently desalted with Sep-Pak tC-18 cartridges (Waters). Desalted samples were dried for 30 min in a SpeedVac vacuum centrifuge and subsequently lyophilized overnight. 10%TFA was used to adjust the pH of the mix to less than 7 before neutral pH fractionation was performed using a 60 min gradient of 4.5 to 45% ACN in 10 mM ammonium formate on an UltiMate 3000 Dual LC nano-HPLC System (Dionex, Thermo Fisher Scientific) equipped with a XBridge Peptide BEH C18 (130 Å, 3.5 μm, 4.6 mm x 250 mm) column (Waters) (flow rate of 1.0 ml / min). Fractions were collected and subsequently pooled in a non-contiguous manner into 8 pools, and lyophilized overnight. Phosphorylated peptides were enriched using TiO2. An aliquot (peptide: TiO2 resin = 1:6) of TiO2 (Titansphere TiO, GL Sciences) was washed twice with 50% methanol and twice with glycolic acid solution (1 M glycolic acid,70% ACN, 3%). Lyophilized peptides were dissolved in glycolic acid solution, mixed with the TiO2 resin, and incubated rotating at RT for 30 min. Samples were loaded onto Mobicol columns (MoBiTec) and shortly centrifuged in a table centrifuge to remove unphosphorylated peptides. Phosphorylated peptides will bind to the TiO2 resin. The resin was washed twice with glycolic acid solution, twice with 200 µl 70%, ACN 3%, TFA and twice with 1% ACN, 0,1% TFA. Phosphorylated peptides were eluted twice using 150 µl 300 mM ammonium hydroxide, eluates were united and immediately acidified with conc. TFA to a pH of 2,5. Samples were desalted using a standard C18 StageTip protocol, dried using a SpeedVac (Eppendorf) and dissolved in 20 ml 2% ACN, 0,1% TFA.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-31", "updatedDate" : "2026-03-04", "submissionDate" : "2026-03-04", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Markus Hartl" ], "labPIs" : [ "Mikolaj Ogrodnik" ], "affiliations" : [ "Ludwig Boltzmann Institute for Traumatology, The Research Center in Cooperation with AUVA, Austrian Cluster for Tissue Regeneration, The laboratory for Tissue Damage Responses in Regeneration and Aging, Donaueschingenstrasse 13, A-1200 Vienna, Austria," ], "instruments" : [ "Orbitrap Exploris 480" ], "softwares" : [ "MaxQuant" ], "quantificationMethods" : [ "TMT" ], "sampleAttributes" : [ "skin", "Sus scrofa domesticus (domestic pig)" ], "organisms" : [ "Sus scrofa domesticus (domestic pig)" ], "organismsPart" : [ "Skin" ], "diseases" : [ ], "references" : [ ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "20221114_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_proteome_poolA.raw", "20221118_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_phospho_Insol.raw", "20221118_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_phospho_poolF.raw", "20221118_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_phospho_poolA.raw", "2022.04_UP000008227_9823_Sus_scrofa_Pig__1protein_per_gene.fasta", "20221118_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_phospho_poolD.raw", "Rscript_Result.zip", "20221114_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_proteome_poolD.raw", "20221118_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_phospho_poolH.raw", "20221114_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_proteome_poolH.raw", "20221114_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_proteome_Insol.raw", "20221114_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_proteome_poolF.raw", "20221114_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_proteome_poolG.raw", "20221118_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_phospho_poolE.raw", "checksum.txt", "20221114_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_proteome_poolB.raw", "MaxQuant_Result_txt.zip", "Raw_file_experiment_list.xlsx", "20221118_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_phospho_poolC.raw", "20221118_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_phospho_poolB.raw", "20221118_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_phospho_poolG.raw", "20221114_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_proteome_poolC.raw", "20221114_E1_RSLC5_Ring_Ogrodnik_MPLGmbH_Academic_proteome_poolE.raw" ], "highlights" : { } }, { "accession" : "PXD075144", "title" : "Metaproteomic profiling reveals viral proteins and associated host proteomic alterations in glioblastoma", "projectDescription" : "Glioblastoma (GB) is a WHO grade 4 brain cancer with dismal prognosis, yet its aetiology remains poorly defined. Although viral involvement has been proposed, findings across studies remain inconsistent, reflecting inherent limitations of individual technologies and cohort size. Here we applied metaproteomic profiling to a publicly available GB proteome dataset (12 control, 21 adjacent, 159 tumour) and an independent cohort of 81 samples (37 control, 44 tumour) to detect viral proteins in tumour and controls tissues. Across cohorts, we detected viral proteins from diverse species, with human herpesviruses (HHV-1, 2, and 8) more frequently detected in GB tumours compared with control tissues. Analysis of the host tumour proteome revealed differential abundance of proteins related to transcriptional regulation, RNA processing, protein translation, immune responses, and mitochondrial-associated metabolism. Correlation analysis identified associations between viral and human proteins, with several linked to biological processes previously implicated in DNA virus-host interactions. Further stratification of tumour by HHV-1 status showed consistent alterations in proteins associated with mitochondrial-associated metabolism, protein turnover, and cell adhesion/signalling. In summary, this study demonstrates the feasibility of metaproteomics for detecting viral components in archival GB tissues. Using this approach, we observed differences in viral protein landscape across cohorts and identified associations between viral presence and host proteomic features, providing a protein-level framework for future studies of virus-host interactions in GB.", "dataProcessingProtocol" : "Protein quantification was performed using DIA-NN (version 1.9.2), based on the spectral library constructed as described above. A double-pass neural network classifier was applied for identification and quantification24. MS1 accuracy, mass accuracy, and scan window width were all set to 0, allowing automatic parameter optimisation. The \"match between runs\" feature was enabled to improve quantification consistency across samples. Precursor-level false discovery rate (FDR) was controlled at 1%. Protein inference was conducted at the protein name level based on the FASTA database. Quantification was performed using the 'quant UMS (high precision)' strategy, and retention time (RT)-dependent cross-run normalisation was applied.", "sampleProcessingProtocol" : "Protein concentrations were determined using Pierce BCA Protein Assays, following the manufacturer's instructions. Proteins were reduced with 25mM dithiothreitol (DTT) (Sigma-Aldrich, D0632-5G) at 56°C for 30 minutes, alkylated with75mM iodoacetamide (IAA) (Sigma-Aldrich, 16125-25G) in the dark at room temperature for 30 minutes and digested overnight at 37°C using sequencing-grade porcine trypsin (Promega, V511A) at a 1:30 enzyme-to-substrate ratio. Resulting peptide mixtures were cleaned using Octadecyl C18 47mm extraction disks (3M, 66883-U) StageTips method", "projectTags" : [ ], "keywords" : [ "Human", "Brain", "Lc-msms", "Glioblastoma", "Tissues" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-11", "updatedDate" : "2026-03-03", "submissionDate" : "2026-03-03", "downloadCount" : 725, "avgDownloadsPerFile" : 7.178218, "botCount" : 19, "hubCount" : 704, "organicCount" : 2, "percentile" : 27, "yearlyDownloads" : [ { "year" : "2026", "count" : 725 } ], "submitters" : [ "Bavani Gunasegaran" ], "labPIs" : [ "Dr Benjamin Heng" ], "affiliations" : [ "Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, NSW, Australia School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, NSW, Australia" ], "instruments" : [ "Orbitrap Exploris 480" ], "softwares" : [ ], "quantificationMethods" : [ "Relative quantification" ], "sampleAttributes" : [ "glioblastoma", "Homo sapiens (Human)", "brain" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Brain" ], "diseases" : [ "Glioblastoma" ], "references" : [ ], "experimentTypes" : [ "Data-independent acquisition" ], "sdrf" : "", "projectFileNames" : [ "20241101_GBM_FFPE_DIA_070.raw", "20241025_GBM_FFPE_DIA_073.raw", "20241025_GBM_FFPE_DIA_004.raw", "20241025_GBM_FFPE_DIA_039.raw", "20241101_GBM_FFPE_DIA_062.raw", "20241025_GBM_FFPE_DIA_103.raw", "checksum.txt", "20241025_GBM_FFPE_DIA_006.raw", "20241025_GBM_FFPE_DIA_049.raw", "20241025_GBM_FFPE_DIA_083.raw", "20241104_GBM_FFPE_DIA_025.raw", "20241101_GBM_FFPE_DIA_011.raw", "20241025_GBM_FFPE_DIA_057.raw", "20241025_GBM_FFPE_DIA_040.raw", "20241025_GBM_FFPE_DIA_037.raw", "20241104_GBM_FFPE_DIA_027.raw", "00d_Bavani_samplelist_DIA.xlsx", "20241101_GBM_FFPE_DIA_069.raw", "20241025_GBM_FFPE_DIA_071.raw", "20241025_GBM_FFPE_DIA_091.raw", "20241104_GBM_FFPE_DIA_031.raw", "20241025_GBM_FFPE_DIA_098.raw", 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"20241104_GBM_FFPE_DIA_029.raw", "20241025_GBM_FFPE_DIA_035.raw", "20241025_GBM_FFPE_DIA_052.raw", "20241101_GBM_FFPE_DIA_066.raw", "20241025_GBM_FFPE_DIA_044.raw", "20241101_GBM_FFPE_DIA_068.raw", "20241025_GBM_FFPE_DIA_087.raw", "20241025_GBM_FFPE_DIA_001.raw", "20241101_GBM_FFPE_DIA_064.raw", "20241101_GBM_FFPE_DIA_017.raw", "20241025_GBM_FFPE_DIA_089.raw", "uniprotkb_Human_AND_model_organism_9606_2024_10_10.fasta", "20241025_GBM_FFPE_DIA_093.raw", "20241101_GBM_FFPE_DIA_021.raw", "20241025_GBM_FFPE_DIA_050.raw", "20241025_GBM_FFPE_DIA_101.raw", "20241025_GBM_FFPE_DIA_033.raw", "20241025_GBM_FFPE_DIA_076.raw", "20241025_GBM_FFPE_DIA_046.raw", "20241025_GBM_FFPE_DIA_003.raw", "20241025_GBM_FFPE_DIA_059.raw" ], "highlights" : { } }, { "accession" : "PXD075159", "title" : "The Carnosine–HNE Michael Adduct as a Redox-Active Species Associated with Nrf2-Dependent Antioxidant and An-ti-Inflammatory Responses", "projectDescription" : "Carnosine (CAR), an endogenous histidine-containing dipeptide, exhibits antioxidant and anti-inflammatory activity in various experimental models; however, its molecular mechanism of action remains poorly understood. Here, we demonstrate that the Michael adduct between CAR and 4-hydroxy-2-nonenal (HNE), which has been detected in previ-ous studies in both in vitro and in vivo settings, mediates its bioactivity, particularly anti-oxidant and anti-inflammatory responses through Nrf2 activation. CAR-HNE adduct was synthesized and its physicochemical, metabolic, and biological properties were evaluated. CAR-HNE exhibited high stability in biological matrices and retained the ability to trans-fer HNE to thiol nucleophiles at a slow rate under physiologically relevant conditions, consistent with electrophile-mediated Nrf2 activation. This kinetic behavior limits the cy-totoxicity typically associated with free HNE while preserving redox signaling capacity. CAR-HNE induced dose-dependent Nrf2 activation and NF-κB inhibition in cell-based assays without the hormetic toxicity observed for free HNE. Mechanistically, CAR-HNE may act as a redox-tunable electrophilic reservoir, restoring nucleophilic tone and modu-lating redox-sensitive transcription factors. In vivo, CAR-HNE attenuated DSS-induced colitis more effectively than equimolar doses of either carnosine or HNE alone. Proteomic analyses revealed modulation of canonical Nrf2-dependent antioxidant pathways. Our findings suggest a conceptual shift in carnosine biology: rather than acting as a classical antioxidant or carbonyl quencher, carnosine functions as a precursor of redox-active elec-trophilic adducts that transduce anti-inflammatory and antioxidant responses via con-trolled RCS signaling.", "dataProcessingProtocol" : "Raw LC–MS/MS files acquired in DDA mode were processed according to a standard label-free quantitative (LFQ) proteomics workflow. Data were analyzed using MaxQuant for peptide/protein identification and LFQ intensity extraction. Spectra were searched against the Mus musculus reference proteome database (target–decoy strategy enabled) to support reliable error estimation. Peptide-spectrum matches and protein identifications were filtered at a 1% false discovery rate (FDR) at both PSM and protein levels. Protein inference was performed using MaxQuant protein grouping, and quantitative values were obtained as LFQ intensities computed with the MaxLFQ algorithm, including across-run normalization. The Match Between Runs feature was enabled to increase identification depth by transferring peptide IDs across LC–MS runs based on accurate mass and aligned retention time. Only proteins supported by unique and/or razor peptides were retained for quantification, while common contaminants, reverse (decoy) entries, and “only identified by site” features were excluded prior to statistical evaluation. Downstream statistical analysis was performed in Perseus using the MaxQuant output tables. LFQ intensities were log2-transformed, and proteins were filtered to retain those quantified in the majority of replicates in at least one condition. Where required for comparative statistics, missing values were imputed (e.g., using a left-shifted normal distribution to model values close to the detection limit). Differential abundance testing was carried out using standard approaches (e.g., two-sample t-test with multiple-testing correction such as Benjamini–Hochberg FDR), accompanied by exploratory multivariate analyses including PCA, hierarchical clustering, and volcano plots. Functional interpretation of the resulting regulated protein sets was subsequently performed using dedicated bioinformatics tools for pathway enrichment and network-based analyses.", "sampleProcessingProtocol" : "Tissue samples were processed using a standardized bottom-up proteomics workflow designed to maximize extraction efficiency and ensure comparability across experimental groups. Briefly, frozen tissue specimens were transferred to dedicated microvials preloaded with zirconia beads and homogenized on a benchtop bead-mill system to achieve rapid and reproducible mechanical disruption. The resulting tissue lysates/homogenates were collected and, where necessary, clarified to remove insoluble debris. Total protein yield was then determined by protein quantification, and equal protein amounts were normalized prior to digestion to minimize technical variability introduced at the sample preparation stage. For proteolytic digestion, proteins were processed using the S-Trap approach, which enables efficient detergent removal and rapid enzymatic digestion. Protein extracts were loaded onto S-Trap micro spin columns following the manufacturer’s recommendations, allowing protein capture within the quartz-fiber matrix and subsequent on-column cleanup. Bound proteins were then digested by on-column trypsinization, generating peptides under controlled conditions that support high digestion efficiency and reduced carryover of matrix components. Following digestion, peptides were eluted from the S-Trap columns and subjected to an additional cleanup/desalting step using C18 ZipTip solid-phase extraction. This step was performed to remove salts and residual contaminants and to concentrate peptide mixtures, thereby improving chromatographic performance and electrospray stability. Purified peptides were analyzed by nano-flow liquid chromatography coupled to high-resolution tandem mass spectrometry (nanoLC–HRMS/MS) using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). Peptide separation was performed under nanoLC conditions prior to ionization by nano-electrospray. The mass spectrometer was operated in data-dependent acquisition (DDA) mode, selecting the top 20 most intense precursor ions detected in each MS1 survey scan for fragmentation and MS/MS acquisition (Top20 method). Raw LC–MS/MS files were subsequently processed through the established bioinformatics pipeline for peptide/protein identification and downstream quantitative analysis.", "projectTags" : [ ], "keywords" : [ "Carnosine; 4-hydroxy-2-nonenal (hne); nrf2 activation; nf-κb; anti-inflammatory; anti-oxidant." ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-05", "updatedDate" : "2026-03-03", "submissionDate" : "2026-03-03", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Alessandra Altomare" ], "labPIs" : [ "Alessandra Altomare" ], "affiliations" : [ "University of MIlan - Department of Pharmaceutical Sciences" ], "instruments" : [ "Orbitrap Fusion" ], "softwares" : [ ], "quantificationMethods" : [ "Relative quantification" ], "sampleAttributes" : [ "colon", "ulcerative colitis", "Mus musculus (Mouse)" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Colon" ], "diseases" : [ "Ulcerative colitis" ], "references" : [ "Altomare A, Baron G, Gado F, Vedova LD, Ferrario G, Davani L, Gilardoni E, Ferrisi R, Mocchetti C, Singh L, Courten B, Carini M, Siracusa R, D'Amico R, Di Paola R, Dallanoce C, Impellizzeri D, Aldini G. The Carnosine-HNE Michael Adduct as a Redox-Active Species Associated with Nrf2-Dependent Antioxidant and Anti-Inflammatory Responses. Antioxidants (Basel). 2026 15(3):388--pubMed:41897532--doi: 10.3390/antiox15030388" ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "22P54-DNBS_6-03.raw", "22P54-DNBS_5-03.raw", "22P54-DNBS_CARM_24-03.raw", "22P54-DNBS_CARM_22-03.raw", "22P54-DNBS_CARM_23-03.raw", "22P54-DNBS_4-03.raw", "22P54-DNBS_3-03.raw", "22P54-DNBS_2-03.raw", "22P54-DNBS_CARM_20-03.raw", "22P54-DNBS_1-03.raw", "22P54-DNBS_4-02.raw", "checksum.txt", "22P54-DNBS_5-02.raw", "22P54-CTRL_29-01.raw", "22P54-DNBS_3-02.raw", "22P54-DNBS_6-02.raw", "22P54-DNBS_1-02.raw", "22P54-DNBS_2-02.raw", "22P54-CTRL_25-01.raw", "22P54-CTRL_26-01.raw", "22P54-CTRL_28-01.raw", "22P54-CTRL_27-01.raw", "22P54-CTRL_25-03.raw", "22P54-DNBS_4-01.raw", "CARMEN_DNBS_VS_DNBS.txt", "22P54-DNBS_2-01.raw", "22P54-DNBS_6-01.raw", "22P54-DNBS_CARM_23-01.raw", "22P54-CTRL_27-02.raw", "22P54-CTRL_29-02.raw", "22P54-CTRL_25-02.raw", "22P54-DNBS_CARM_23-02.raw", "22P54-CTRL_30-02.raw", "22P54-CTRL_30-03.raw", "22P54-CTRL_29-03.raw", "22P54-CTRL_27-03.raw", "22P54-CTRL_30-01.raw", "22P54-DNBS_CARM_19-03.raw", "DNBS_VS_CTR.txt", "22P54-DNBS_CARM_20-01.raw", "22P54-CTRL_26-03.raw", "22P54-DNBS_3-01.raw", "22P54-DNBS_5-01.raw", "22P54-DNBS_CARM_22-01.raw", "22P54-DNBS_1-01.raw", "22P54-CTRL_28-02.raw", "22P54-DNBS_CARM_24-01.raw", "22P54-CTRL_26-02.raw", "22P54-DNBS_CARM_19-01.raw", "CARMEN_DNBS_VS_CTR.txt", "22P54-DNBS_CARM_22-02.raw", "22P54-DNBS_CARM_20-02.raw", "22P54-DNBS_CARM_24-02.raw", "22P54-CTRL_28-03.raw", "22P54-DNBS_CARM_19-02.raw" ], "highlights" : { } }, { "accession" : "PXD075165", "title" : "Alpibectir–Ethionamide combination (AlpE) for the treatment of tuberculosis", "projectDescription" : "MS/MS data for profiling of Mycobacterium tuberculosis treated with Compound 1, Compound 2, Alpibectir, Compound 3 and vehicle control: 10 samples were analyzed in parallel (TMT 10-plex) to generate whole proteome profiling data. Mycobacterium tuberculosis were treated for 72h with 1µM of the respective compound: Samples 1 and 2 treated with Compound 1, samples 3 and 4 with Compound 2, samples 5 and 6 with Alpibectir, samples 7 and 8 with Compound 3 and samples 9 and 10 with DMSO (vehicle control experiments n=1 and n=2).", "dataProcessingProtocol" : "Mascot 2.4 (Matrix Science, Boston, MA) was used for protein identification by using a 10 parts per million mass tolerance for peptide precursors and 20 mD (HCD) mass tolerance for fragment ions. To create the fasta file for mascot searching, all proteins corresponding to the taxonomy ‘Mycobacterium tuberculosis H37Rv’ were downloaded from Uniprot (release 20170621) and supplemented with common contaminant protein sequences of bovine serum albumin, porcine trypsin and mouse, rat, sheep and dog keratins. To assess the false discovery rate (FDR) “decoy” proteins (reverse of all protein sequences) were created and added to the database, resulting in a database containing a total of 7994 protein sequences, 50% forward, 50% reverse. Unless stated otherwise, we accepted protein identifications as follows: (i) For single spectrum to sequence assignments, we required this assignment to be the best match and a minimum Mascot score of 31 and a 10× difference of this assignment over the next best assignment. Based on these criteria, the decoy search results indicated <1% false discovery rate (FDR). (ii) For multiple spectra to sequence assignments and using the same parameters, the decoy search results indicated <0.1% FDR. Quantified proteins were required to contain at least 2 unique peptide matches. FDR for quantified proteins was < 0.1%. The raw data table for the Whole Proteome Profiling experiment can be found in Supplementary Table 1.", "sampleProcessingProtocol" : "37.5 µl of Mycobacterium tuberculosis protein extract were combined with SDS sample buffer and the proteins were digested according to a modified single pot solid-phase sample preparation (SP3) protocol (Ref. 2, 3). Briefly, proteins were bound to paramagnetic beads (SeraMag Speed beads, GE Healthcare, CAT#45152105050250, CAT#651521050502) in 50% Ethanol (final concentration). Beads were washed 4 times with 70 % Ethanol and proteins digested by resuspending in 0.1 mM HEPES (pH 8.5) containing TCEP, Chloroacetamide, Trypsin and LysC followed by o/n incubation. Peptides were labeled with isobaric mass tags (TMT10, Thermo FisherScientific, Waltham, MA using the 10-plex TMT reagents, enabling relative quantification of 10 conditions in a single experiment (Ref. 1, 4). The labeling reaction was performed in 40 mM triethylammoniumbicarbonate, pH 8.5 at 22°C and quenched with glycine. Labeled peptide extracts were combined into a single sample per experiment, lyophilized and subjected to LC-MS analysis. Samples were pre-fractionated by reversed-phase chromatography at high pH to 16 samples of which 10 were measured (Ref. 5). After lyophilization, dried samples were resuspended in 0.05 % trifluoroacetic acid in water. Half of the sample was injected into an Ultimate3000 nanoRLSC (Dionex) coupled to a Q-Exactive (Thermo Fisher Scientific). Peptides were separated into custom-made 35 cm x 100 um (ID) reversed-phase columns (Reprosil) at 55°C, gradient elution was performed from 3.5 % acetonitrile to 29 % acetonitrile in 0.1 % formic acid and 3.5 % DMSO over 120 minutes. The Q Exactive Plus operated with data-dependent top 10 method. MS spectra were acquired using 70.000 resolution and an ion target of 3E6. Higher energy collisional dissociation (HCD) scans were performed with 35 % NCE at 35.000 resolution (at m/z 200), and ion target settings 1. Werner, T., Sweetman, G., Savitski, M.F., Mathieson, T., Bantscheff, M., and Savitski, M.M. (2014). Ion Coalescence of Neutron Encoded TMT 10-Plex Reporter Ions. Anal. Chem. 86, 3594–3601 2. Hughes CS, Foehr S, Garfield DA, Furlong EE, Steinmetz LM, Krijgsveld J. Ultrasensitive proteome analysis using paramagnetic bead technology. Mol Syst Biol. 2014 Oct 30;10:757 3. Moggridge S, Sorensen PH, Morin GB, Hughes CS. Extending the Compatibility of the SP3 Paramagnetic Bead Processing Approach for Proteomics. J. Proteome Res. 17, 1730–1740 (2018). 4. Werner, T., Becher, I., Sweetman, G., Doce, C., Savitski, M.M., and Bantscheff, M. (2012). High-Resolution Enabled TMT 8-plexing. Anal. Chem. 84, 7188–7194", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-02", "updatedDate" : "2026-03-03", "submissionDate" : "2026-03-03", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Sascha Knecht" ], "labPIs" : [ "Thilo Werner" ], "affiliations" : [ "Target Discovery, GSK / Cellzome" ], "instruments" : [ "Q Exactive" ], "softwares" : [ ], "quantificationMethods" : [ "TMT" ], "sampleAttributes" : [ "Mycobacterium tuberculosis", "Bacteria", "Mycobacterium tuberculosis H37Rv" ], "organisms" : [ "Mycobacterium tuberculosis", "Bacteria", "Mycobacterium tuberculosis h37rv" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.1038/S41467-026-71460-6" ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "WPSettings.dat", "F405168.dat", "02495_F1_R1_P0200208C04_TMT10.raw.rar", "F405161.dat", "02495_F1_R1_P0200208C05_TMT10.raw.rar", "02495_F1_R1_P0200208C09_TMT10.raw.rar", "F405163.dat", "F405166.dat", "02495_F1_R1_P0200208C06_TMT10.raw.rar", "IndexerVolumeGuid", "02495_F1_R1_P0200208C08_TMT10.raw.rar", "F405170.dat", "02495_F1_R1_P0200208C07_TMT10.raw.rar", "checksum.txt", "F405167.dat", "F405164.dat", "02495_F1_R1_P0200208C01_TMT10.raw.rar", "02495_F1_R1_P0200208C10_TMT10.raw.rar", "F405162.dat", "F405165.dat", "02495_F1_R1_P0200208C03_TMT10.raw.rar", "F405169.dat", "02495_F1_R1_P0200208C02_TMT10.raw.rar" ], "highlights" : { } }, { "accession" : "PXD075103", "title" : "Peptidomics-guided identification of TRPV1-targeting peptides from Apis mellifera venom and macrophage proteomics", "projectDescription" : "Bee venom has been traditionally used in the treatment of inflammatory disorders and is known to contain diverse bioactive peptide components. In this study, we systematically characterized the Apis mellifera venom peptidome and investigated peptides enriched under TRPV1 pull-down conditions with potential inflammatory modulatory activity. Crude venom was fractionated by AKTA chromatography, and individual fractions were evaluated by SDS–PAGE. Protein bands were subjected to in-gel digestion followed by LC–MS/MS protein identification. Fraction F4, which displayed a prominent A280 absorbance peak during AKTA chromatography, was further analyzed by LC–MS/MS–based peptidomics. De novo sequencing results from fraction F4 were used to construct an in-house custom peptide database. Two independent GST pull-down workflows (exploratory and in-house) were performed using GST-TRPV1 and GST controls. Candidate peptides enriched in the GST-TRPV1 pull-down relative to GST controls were identified following background subtraction and peak-area filtering criteria. Selected synthetic peptide candidates were subsequently evaluated using macrophage-based proteomics experiments to assess their potential inflammatory modulatory effects. The deposited dataset includes raw LC–MS/MS files and processed outputs for gel-based protein identification, venom peptidomics, pull-down–associated peptide identification, macrophage proteomics (MaxQuant analysis), and the custom venom peptide FASTA library used for database searches.", "dataProcessingProtocol" : "For gel-based protein identification, raw MS and MS/MS spectra were processed using Proteome Discoverer (v2.4, Thermo Fisher Scientific) and searched against the Apis FASTA database downloaded from UniProtKB (taxonomy ID: 7459; 98,839 entries; accessed on October 10, 2023). For peptidomic analyses, raw MS/MS files were processed using PEAKS X Pro (PEAKS Studio 10.6; Bioinformatics Solutions Inc., Waterloo, Canada) using a de novo sequencing workflow. The precursor mass tolerance was set to 20 ppm and fragment ion tolerance to 0.02 Da. No enzyme specificity was applied. Variable modifications included oxidation, deamidation, and protein N-terminal acetylation, allowing up to three variable post-translational modifications per peptide. Peptide sequences with ALC ≥ 50% were deposited and used to construct an in-house Apis mellifera venom peptide database. Peptides with ALC ≥ 80% and non-zero peak area were considered high-confidence endogenous peptides. For GST pull-down experiments, candidate TRPV1-associated peptides were identified using PEAKS de novo sequencing and the PEAKS DB algorithm against the in-house Apis mellifera venom peptide database described above. For macrophage proteomic analysis, protein identification and label-free quantification (LFQ) were performed using MaxQuant (version 2.6.5.0). The reference protein database was retrieved from the reviewed UniProt Mouse proteome (taxonomy ID: 10088; 17,285 entries; accessed on September 4, 2024). Trypsin was specified as the digestion enzyme, allowing up to two missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine and protein N-terminal acetylation were set as variable modifications. A false discovery rate (FDR) of 1% was applied at both peptide and protein levels. The MaxQuant output file (MacrophageProteomics_proteinGroups.txt) contains all experimental conditions analyzed in the MaxQuant project. For analyses reported in the associated manuscript, dexamethasone- and peptide P4–treated samples were not included. Corresponding columns were excluded, and proteins detected exclusively in those samples were removed. The manuscript-specific dataset is provided as MacrophageProteomics_proteinGroups_filtered_NoDXMSNoP4.xlsx.", "sampleProcessingProtocol" : "Crude Apis mellifera venom was fractionated by AKTA chromatography. Individual fractions were analyzed by SDS–PAGE, and visible protein bands were excised, destained, reduced, alkylated, and subjected to in-gel enzymatic digestion prior to LC–MS/MS analysis for protein identification. Protein identification was performed using an Orbitrap Fusion mass spectrometer coupled to an EASY-nLC 1000 system (Thermo Fisher Scientific). Peptides were separated on a C18 column (Acclaim PepMap, 100 μm id × 2 cm, 3 μm particle size) using a 60 min linear gradient at 300 nL/min with 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). Fraction F4 was subjected to 3 kDa ultrafiltration (Millipore) to enrich low-molecular-weight peptides (<3 kDa), followed by C18 solid-phase extraction for desalting and concentration. The resulting peptide samples were analyzed by nano LC–MS/MS under the same instrumental conditions for peptidomics characterization. For GST pull-down experiments, purified GST-TRPV1 or GST control proteins were immobilized on glutathione resin, incubated with fraction F4 peptide samples (after concentration and salt removal), washed, and eluted. The eluted samples were further purified, filtered, concentrated, and analyzed by nano LC–MS/MS under the same instrumental conditions. Macrophage proteomic profiling was performed using an Orbitrap Exploris 480 mass spectrometer coupled with an EASY-nanoLC 1200 system (Thermo Fisher Scientific). Peptides were separated on a 75 μm × 20 cm C18 analytical column using a 60 min gradient at 300 nL/min. Raw data were acquired using Thermo Xcalibur software.", "projectTags" : [ ], "keywords" : [ "Apis mellifera venom", "Proteomics", "Trpv1", "Inflammation", "Peptidomics", "Pull-down", "Macrophage" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-17", "updatedDate" : "2026-03-02", "submissionDate" : "2026-03-02", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Kai Wang" ], "labPIs" : [ "Hongcheng Zhang" ], "affiliations" : [ "State Key Laboratory of Resource Insects, Institute of Apicultural Research, Chinese Academy of Agricultural Sciences" ], "instruments" : [ "Orbitrap Fusion", "Orbitrap Exploris 480" ], "softwares" : [ ], "quantificationMethods" : [ "label-free quantification" ], "sampleAttributes" : [ "venom", "Apis mellifera (Honeybee)", "macrophage", "Mus musculus (Mouse)" ], "organisms" : [ "Mus musculus (mouse)", "Apis mellifera (honeybee)" ], "organismsPart" : [ "Venom", "Macrophage" ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.1021/ACS.JNATPROD.5C01554" ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "Inhouse_GSTTRPV1_pullDown_proteins.csv", "GelBandProteinID_Am_F4_PD.xlsx", "MacrophageProteomics_proteinGroups_filtered_NoDXMSNoP4.xlsx", "MacrophageProteomics_P1_rep3.raw", "MacrophageProteomics_DXMS_rep2.raw", "Exploratory_GST_pullDown.raw", "GelBandProteinID_Am_F3.raw", "MacrophageProteomics_Control_rep3.raw", "MacrophageProteomics_LPS_rep2.raw", "Peptidomics_Am_F4.raw", "MacrophageProteomics_P3_rep2.raw", "Exploratory_GST_pullDown_proteins.csv", "MacrophageProteomics_CPS_rep1.raw", "checksum.txt", "MacrophageProteomics_P3_rep3.raw", "Inhouse_GST_pullDown.raw", "MacrophageProteomics_P2_rep1.raw", "Peptidomics_Am_F4_PEAKS.csv", "MacrophageProteomics_DXMS_rep3.raw", "MacrophageProteomics_Control_rep2.raw", "Exploratory_GSTTRPV1_pullDown.raw", "GelBandProteinID_Am_F3_PD.xlsx", "MacrophageProteomics_P2_rep2.raw", "MacrophageProteomics_LPS_rep3.raw", "ApisMellifera_Venom_denovoPeptideLibrary.fasta", "MacrophageProteomics_P4_rep1.raw", "MacrophageProteomics_P1_rep1.raw", "MacrophageProteomics_CPS_rep2.raw", "MacrophageProteomics_Control_rep1.raw", "Exploratory_GST_pullDown_protein_peptides.csv", "Inhouse_GSTTRPV1_pullDown_protein_peptides.csv", "Inhouse_GSTTRPV1_pullDown.raw", "Inhouse_GST_pullDown_proteins.csv", "MacrophageProteomics_P2_rep3.raw", "MacrophageProteomics_CPS_rep3.raw", "Exploratory_GSTTRPV1_pullDown_proteins.csv", "Exploratory_GSTTRPV1_pullDown_protein_peptides.csv", "MacrophageProteomics_P4_rep2.raw", "Inhouse_GST_pullDown_protein_peptides.csv", "MacrophageProteomics_P1_rep2.raw", "MacrophageProteomics_DXMS_rep1.raw", "MacrophageProteomics_P3_rep1.raw", "GelBandProteinID_Am_F4.raw", "MacrophageProteomics_P4_rep3.raw", "MacrophageProteomics_LPS_rep1.raw", "MacrophageProteomics_proteinGroups.txt" ], "highlights" : { } }, { "accession" : "PXD075028", "title" : "Landscape and dynamics of TadA-dependent RNA editing in Escherichia coli reveal a role in nutrient-rich growth", "projectDescription" : "Adenosine-to-inosine A-to-I mRNA editing alters genetic information post-transcriptionally and can impact protein sequence and function, yet its regulation in bacteria remains unclear. Here, we profiled A-to-I editing in Escherichia coli across nutrient-rich LB and minimal M9 media and different growth phases. Our analysis expanded the repertoire of TadA-dependent A-to-I edited mRNAs to 27, including 12 novel sites, and revealed that editing levels were dynamic and markedly increased at stationary phase in LB but not in M9. Editing levels were independent of mRNA expression yet correlated with tRNA-Arg2 downregulation, and overexpressing tRNA-Arg2 reduced mRNA editing, demonstrating substrate competition for TadA, the sole bacterial tRNA adenosine deaminase. Mutants with TadA-deficient editing or reduced tRNA-Arg2 expression displayed similar LB-specific growth defects. Moreover, tRNA-Arg2 expression, tRNA-Arg2-dependent codon usage, and tRNA-Arg2 editing were all elevated in LB compared to M9. These findings establish regulatory principles for bacterial RNA editing, implicate tRNA editing in nutrient-responsive fitness, and provide a framework to explore the physiological roles of mRNA editing", "dataProcessingProtocol" : "The mass spectrometry data was analyzed using the DIA-NN software version 2.2 (Demichev et al. 2020; Messner et al. 2021). The tryptic searching against Escherichia coli strain K12_UP000000625_83333 from Feb 2026 (4403 entries), with minimal peptide length set to 7, maximum number of missed cleavages set to 1, cysteine carbamidomethylation enabled as a fixed modification, and protein N-term acetylation and Oxidation on methionine enabled as a variable modification. Peptide- and protein-level false discovery rates (FDRs) were filtered to 1%.  ", "sampleProcessingProtocol" : "Proteolysis Bacteria cells were lyzed in 8.5M Urea, 400mM ammonium bicarbonate and 10mM DTT, sonicated twice (90%, 10-10, 5’), and centrifuged (10,000g, 10’). Protein amount was estimated using Bradford readings. The samples were reduced (60ºC for 30 min), modified with 35.2mM iodoacetamide in 100mMammonium bicarbonate (room temperature for 30 min in the dark) and digested in 1.5M Urea, 66mM ammonium bicarbonate with modified trypsin (Promega), overnight at 37oC in a 1:50 (M/M) enzyme-to-substrate ratio. An additional second digestion with Trypsin was done for 4 hours at 37oC in a 1:100 (M/M) enzyme-to-substrate ratio. The tryptic peptides were desalted using Oasis HLB 96-well µElution Plate (Waters) dried and re-suspended in 0.1% Formic acid in 2% acetonitrile. Mass spectrometry analysis The resulting peptides were analyzed by LC-MS/MS using an Exploris 480 mass spectrometer (Thermo) fitted with a capillary UHPLC (Vanquish Neo, Thermo scientific). The peptides were loaded in solvent A (0.1% formic acid in water) on an C18 reversed phase analytical column (Ionoptics, AUR3-25075C18-XT, 25cm x 75um ID, 1.7um) The peptides mixture was resolved with a 6 to 34% linear gradient of solvent B (80% acetonitrile with 0.1% formic acid in water) for 120 minutesfollowed by gradient of 0.1min increase of 34 to 99% and 14 minutes at 99% solvent B at flow rates of 0.15 μl/min. Mass spectrometry was performed in a positive mode using repetitively full MS scan (m/z 380–985, resolution 120,000) followed by DIA scans (10Da isolation windows with 1 m/z overlap, and resolution 30,000).", "projectTags" : [ ], "keywords" : [ "Nutrient-rich growth", "Tada-dependent rna editing" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-24", "updatedDate" : "2026-02-28", "submissionDate" : "2026-02-28", "downloadCount" : 4, "avgDownloadsPerFile" : 0.44444445, "botCount" : 0, "hubCount" : 4, "organicCount" : 0, "percentile" : 0, "yearlyDownloads" : [ { "year" : "2026", "count" : 4 } ], "submitters" : [ "Dganit Melamed Kadosh" ], "labPIs" : [ "Dan Bar-Yaacov" ], "affiliations" : [ "1The Shraga Segal Department of Microbiology, Immunology, and Genetics, Ben-Gurion University of the Negev, Israel" ], "instruments" : [ "Orbitrap Exploris 480" ], "softwares" : [ ], "quantificationMethods" : [ "label-free quantification" ], "sampleAttributes" : [ "Bacteria", "Escherichia coli (strain K12)" ], "organisms" : [ "Bacteria", "Escherichia coli (strain k12)" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.1128/MBIO.00551-26" ], "experimentTypes" : [ "Data-independent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "Seq97397_DIA_480Ex2.raw", "checksum.txt", "Seq97392_DIA_480Ex2.raw", "Sample_list.xlsx", "Seq97396_DIA_480Ex2.raw", "report_97392_7.zip", "Seq97395_DIA_480Ex2.raw", "Seq97394_DIA_480Ex2.raw", "Seq97393_DIA_480Ex2.raw" ], "highlights" : { } }, { "accession" : "PXD075040", "title" : "Genetic and pharmacological inactivation of peptidoglycan remodeling increases antibiotic susceptibility of vancomycin-resistant Enterococcus faecium", "projectDescription" : "Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of healthcare-associated infections globally and demands new approaches for treatment. Here we show that genetic and pharmacological inactivation of a highly conserved NlpC/P60 peptidoglycan hydrolase, secreted antigen A (SagA), enhanced vancomycin susceptibility of VREfm ex vivo and in vivo. Notably, genetic deletion of sagA impaired VREfm peptidoglycan remodeling, growth and increased the activity of vancomycin. We then identified first-in-class covalent NlpC/P60 peptidoglycan hydrolase inhibitors and demonstrated that pharmacological inactivation of SagA activity also impaired peptidoglycan remodeling and increased the efficacy of vancomycin in several VREfm clinical isolates. Our study reveals peptidoglycan hydrolases are druggable targets whose inactivation improves the efficacy of vancomycin against VREfm.", "dataProcessingProtocol" : "RAW file uploaded here were further analyzed as previously described (Njomen E. et al. Nat. Chem. 2024) with small modifications regarding the proteome dataset Enterococcus faecium ERV165 UniProt database (UP000005678).", "sampleProcessingProtocol" : "The protocol was adapted from Njomen E. et al. Nat. Chem. 2024. VREfm ERV165 were grown from overnight culture in 15 mL of fresh BHI till OD600~0.6. Bacteria were then centrifuged and resuspended in 1 mL of BHI (OD600~9), followed by incubation with pghi-4 (10, 25 or 50 μM) for 1 hour at 37°C and 200 RPM shaking. Bacterial pellets were collected by centrifugation (4,800 × g for 10 min), washed twice with PBS and immediately processed or stored at −80 °C. Bacterial pellets were resuspended in 360 μL PBS and lysed with 0.1 mm glass beads (BioSpec) using FastPrep system (MP Biomedicals, settings: 6 m/s, 2 cycles, 45 s for each cycle). Proteins were quantified by Pierce™ BCA Protein Assay (ThermoFisher) and normalized to 2 mg/mL. 1 mg/0.5 mL of bacterial proteome was incubated with iodoacetamide-desthiobiotin (IA-DTB, 100 μM) for 1 hour at room temperature agitating. Bacterial proteins were precipitated by cold methanol (600 μL), chloroform (200 μL), and water (100 μL), followed by vortexing and centrifugation at 16,000 × g for 10 min at 4°C. Liquids were carefully aspirated, proteins were washed with cold methanol, pelleted by centrifugation (16,000 × g for 10 min at 4°C) and air-dried for 5 min. Protein pellets were resuspended in 90 μL of buffer (9 M urea, 10 mM DTT, 50 mM triethylammonium bicarbonate (TEAB) pH 8.5), heated at 65°C for 20 min, followed by treatment with iodoacetamide (50 mM) for 30 min at 37°C. The insoluble residues were pelleted by centrifugation and clear solutions were sonicated. Samples were diluted with 300 μL TEAB buffer and trypsinized (5 μL of 0.4 μg/μL trypsin in trypsin buffer supplemented with 25 mM CaCl2) overnight at 37°C. Samples were treated with 400 µL of wash buffer (50 mM TEAB, 150 mM NaCl, 0.2% NP-40) containing 50 µL of streptavidin agarose to the peptide samples, followed by rotation at room temp for 2 hours. Suspensions were briefly centrifuged, and beads-containing suspensions were loaded on BioSpin columns. The beads were sequentially washed with 3 × 1 mL wash buffer, 3 × 1 mL PBS, 3 × 1 mL MiliQ water. Beads-bound peptides were eluted by addition of 2 × 200 µL of 80% acetonitrile (0.1% formic acid) and the eluate was concentrated by SpeedVac. Peptides were resuspended in 70 μL EPPS buffer (200 mM, pH 8.0), supplemented with 30% acetonitrile, vortexed and sonicated for 5 min. Peptides were tandem mass tag (TMT)-labeled by adding 3 μL of 10 mg/mL TMT10plex tag and incubating for 1 hour at room temperature. The reaction was quenched by sequential addition of hydroxylamine (3 μL of a 5% aqueous solution, 15 min at room temperature) and formic acid (5 μL), followed by concentration using SpeedVac. Samples were resuspended in 500 μL Velos buffer A (95% water, 5% acetonitrile, 0.1% formic acid), acidified with 20 μL of formic acid, desalted (Sep-Pak C18 Cartridge) and concentrated by SpeedVac. Desalted and concentrated samples were redissolved in 500 μL Velos buffer A and HPLC-fractionated. Fractionation and TMT LC-MS analysis was followed as previously described (Njomen E. et al. Nat. Chem. 2024).", "projectTags" : [ ], "keywords" : [ "Chemoproteomics", "Peptidoglycan hydrolase", "Antibiotic adjuvant", "Vancomycin-resistant enterococcus faecium", "Covalent inhibitor" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-13", "updatedDate" : "2026-02-28", "submissionDate" : "2026-02-28", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Kyong Fam" ], "labPIs" : [ "Howard C. Hang" ], "affiliations" : [ "Department of Immunology & Microbiology, Scripps Research, USA" ], "instruments" : [ "Orbitrap Fusion" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Enterococcus faecium ERV165" ], "organisms" : [ "Enterococcus faecium erv165" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ "Fam KT, Chodisetti PK, Wang Z, Homer JA, Smedley CJ, Kitamura S, Silva B, Xiong Y, Hansel-Harris A, Holcomb M, Babarinde S, Turner AM, Van Tyne D, Wilson IA, Forli S, Cravatt BF, Park D, Wolan DW, Moses JE, Hang HC. Genetic and pharmacological inactivation of peptidoglycan remodeling increases antibiotic susceptibility of vancomycin-resistant <i>Enterococcus faecium</i>. bioRxiv. 2026:2025.09.11.675460--pubMed:41889990--doi: 10.1101/2025.09.11.675460" ], "experimentTypes" : [ "Top-down proteomics" ], "sdrf" : "", "projectFileNames" : [ "20231009_SoL_ERV165_YX_pghi-4_02.raw", "20231009_SoL_ERV165_YX_pghi-4_10.raw", "20231009_SoL_ERV165_YX_pghi-4_04.raw", "20231009_SoL_ERV165_YX_pghi-4_09.raw", "20231009_SoL_ERV165_YX_pghi-4_07.raw", "20231009_SoL_ERV165_YX_pghi-4_12.raw", "20231009_SoL_ERV165_YX_pghi-4_05.raw", "20231009_SoL_ERV165_YX_pghi-4_01.raw", "20231009_SoL_ERV165_YX_pghi-4_08.raw", "checksum.txt", "20231009_SoL_ERV165_YX_pghi-4_03.raw", "20231009_SoL_ERV165_YX_pghi-4_06.raw", "20231009_SoL_ERV165_YX_pghi-4.txt", "20231009_SoL_ERV165_YX_pghi-4_11.raw" ], "highlights" : { } }, { "accession" : "PXD074970", "title" : "Proteomic screen of PACS1 interactors in HCT116 cells", "projectDescription" : "PACS1 syndrome is a neurodevelopmental disorder caused by a recurrent heterozygous missense mutation in PACS1 (p.R203W). We previously showed that PACS1R203W aberrantly potentiates HDAC6 activity, leading to Golgi fragmentation and neuronal deficits through an unresolved mechanism. To elucidate how PACS1R203W and HDAC6 disrupt Golgi positioning, we performed a proteomic screen to identify additional PACS1R203W partners involved in microtubule-based trafficking. HCT116 cells expressing FLAG-tagged PACS1R203W or PACS1 (control) underwent anti-FLAG immunoprecipitation (IP), and the co-purified proteins were analyzed by tandem mass spectrometry (LC-MS/MS).", "dataProcessingProtocol" : "Protein identification was achieved by analyzing each gel section’s tandem mass spectrometry data using Sciex ProteinPilot software (version 4.5, revision 1656). This software employs the Paragon algorithm (version 4.5.0.0, revision 1654), which searches against a Human UniProt Fasta database (captured on March 15, 2019), supplemented with common contaminating proteins (e.g., human keratins, porcine trypsin, and bovine albumin). The ProteinPilot search parameters included typical biological modifications (e.g., Met oxidation, N-terminal Gln conversion to pyroGlu, and acetylation) as variable modifications, with carboxyamidomethylation of cysteine as a fixed modification to account for the reduction and alkylation steps prior to trypsin digestion. Protein identifications are reported for proteins that matched at least two peptides at >99% confidence (<1% FDR) compared to an inverse decoy database.", "sampleProcessingProtocol" : "HCT116 cells were transfected with vector (pcDNA3), PACS1-flag or PACS1 R203W-flag. After 24 h, cells were lysed in lysis buffer(50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% DOC and 2 µM ATP) containing protease inhibitors (0.5 mM PMSF, 0.25 mM Pefabloc, 0.1 mM Aprotinin, 3 μM E-64 and 0.1 mM Leupeptin) and phosphatase inhibitors (1 mM Na3VO4 and 20 mM NaF). The lysates were precleared at 16,000 × g for 15 min, and 10% of the supernatant was mixed with 5x Laemmli sample buffer for input controls. The remaining lysate was incubated with a 50% slurry of anti-FLAG M2 affinity beads at 4°C for 2 h. The beads were washed three times in the same buffer used for lysis, and the captured proteins were eluted in 2x Laemmli sample buffer at 95°C for 5 min. The gels were fixed and stained with a Pierce Silver Stain for Mass Spectrometry. Proteins within the regions of interest on the gels were excised, reduced with dithiothreitol (DTT), alkylated with iodoacetamide, digested with trypsin, and dried using a SpeedVac concentrator. The dried samples were reconstituted in 0.1% formic acid and applied to an Eksigent nanoflow LC system interfaced with a Sciex 5600+ TripleTOF mass spectrometer, except that a gradient from 5% to 40% acetonitrile was used over 30 min.", "projectTags" : [ ], "keywords" : [ "Pacs1", "Pacs1 syndrome" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-05", "updatedDate" : "2026-02-27", "submissionDate" : "2026-02-27", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Gary Thomas" ], "labPIs" : [ "Gary Thomas" ], "affiliations" : [ "University of Pittsburgh" ], "instruments" : [ "TripleTOF 5600" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Homo sapiens (Human)" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ "Yang Y, Thomas L, Chen K, Villar-Pazos S, Haffey WD, D'Agostino A, Fanelli K, Choi YJ, Rathi V, Matlapudi MS, Greis KD, Thomas G. PACS1 syndrome mutation disrupts dynein-mediated cargo transport via HDAC6 and BICD2. Commun Biol. 2026 9(1):450--pubMed:41888583--doi: 10.1038/s42003-026-09924-0" ], "experimentTypes" : [ "Top-down proteomics" ], "sdrf" : "", "projectFileNames" : [ "TQ1_080219_12_PACS1_WT_section_5_all_entries.group", "TQ1_080219_14_PACS1_R203W_section_6_all_entries.group", "TQ1_080219_14_PACS1_R203W_section_6_all_entries.mgf", "TQ1_080219_08_PACS1_R203W_section_3_all_entries.group", "TQ1_080219_06_PACS1_WT_section_2_all_entries.mgf", "TQ1_080219_10_pcDNA_section_4_all_entries.group", "TQ1_080219_06_PACS1_WT_section_2_all_entries.wiff.scan", "TQ1_080219_08_PACS1_R203W_section_3_all_entries.wiff", "TQ1_080219_14_PACS1_R203W_section_6_all_entries.wiff", "checksum.txt", "TQ1_080219_06_PACS1_WT_section_2_all_entries.group", "TQ1_080219_12_PACS1_WT_section_5_all_entries.wiff", "TQ1_080219_14_PACS1_R203W_section_6_all_entries.wiff.scan", "TQ1_080219_08_PACS1_R203W_section_3_all_entries.wiff.scan", "TQ1_080219_06_PACS1_WT_section_2_all_entries.wiff", "TQ1_080219_10_pcDNA_section_4_all_entries.wiff", "TQ1_080219_03_pcDNA_section_1_all_entries.wiff.scan", "TQ1_080219_10_pcDNA_section_4_all_entries.mgf", "TQ1_080219_03_pcDNA_section_1_all_entries.group", "TQ1_080219_12_PACS1_WT_section_5_all_entries.wiff.scan", "TQ1_080219_03_pcDNA_section_1_all_entries.wiff", "TQ1_080219_03_pcDNA_section_1_all_entries.mgf", "TQ1_080219_12_PACS1_WT_section_5_all_entries.mgf", "TQ1_080219_10_pcDNA_section_4_all_entries.wiff.scan", "TQ1_080219_08_PACS1_R203W_section_3_all_entries.mgf" ], "highlights" : { } }, { "accession" : "PXD074944", "title" : "β2 adrenergic receptors orchestrate neutrophil demargination and recruitment to the ischemic heart following myocardial infarction- 2nd mass spectrometry study", "projectDescription" : "Neutrophils play a crucial role in instigating inflammation as well as its resolution postmyocardial infarction (MI). Although granulopoiesis in the bone marrow (BM) is the major source of cardiac neutrophils post-MI, infiltration of neutrophils to the heart occurs much quicker than peak granulopoiesis. Using a combination of flow cytometry, BM ablation of hematopoietic stem cells, confocal microscopy and multiple proteomics analysis, we found that the first wave of neutrophils recruited to the ischemic heart is exclusively sourced from vasculature and not from granulopoiesis in the BM/ spleen. The MIevoked neutrophilia during the early hours bore all hallmarks of demargination induced by classical demarginating agents such as dexamethasone/ norepinephrine (NE). Various pharmacological and genetic strategies aimed at suppressing NE synthesis or disruption of β-AR signaling reduced both neutrophil demargination as well as recruitment to the heart. Interestingly, however, despite a marked reduction in cardiac neutrophil burden only short-term inhibition of β-ARs improved cardiac remodeling and function. Our findings support a pharmacological strategy to contain the initial onslaught of neutrophils on the ischemic heart using β2-AR blockers to regulate the otherwise runaway inflammatory response.", "dataProcessingProtocol" : "The raw files were searched using Mascot Daemon by Matrix Science version 2.5.1 (Matrix Science, Boston, MA) via ProteomeDiscoverer (Thermo) against the database provided by the user. The mass accuracy of the precursor ions was set to 10 ppm, accidental pick of 113C peaks was also included into the search. The fragment mass tolerance was set to 0.5 Da. Carbamidomethylation (Cys) is used as a fixed modification and considered variable modifications were oxidation (Met) and deamidation (N and Q). Four missed cleavages for the enzyme were permitted. A decoy database was also searched to determine the false discovery rate (FDR) and peptides were filtered according at 1% FDR. Proteins identified with at least two unique peptides were considered as reliable identification. Any modified peptides are manually checked for validation. Label free quantitation was performed using the spectral count approach, in which the relative protein quantitation is measured by comparing the number of MS/MS spectra identified from the same protein in each of the multiple LC/MSMS datasets. Scaffold (Proteome Software, Portland, OR) was used for data analysis. Student-t test was performed by scaffold to evaluate if the folder change for certain proteins is significant (p<0.05).", "sampleProcessingProtocol" : "Cell pellets were washed with PBS three times before resuspend in 100 µL of 5% SDS buffer in 50 mM TEAB (Triethylammonium bicarbonate) solution. Samples then were vortexed briefly before proceeding to sonication by using the Bioruptor® Pico (Diagenode, Denville, NJ) following manufacturer’s suggested protocol. Briefly, sonication temperature was set at 4C; sonication cycle was set at 30 sec on and 30 sec off. A total of 10 cycles were done for the cell pellets. (Sometimes, DNAs are not sheared well in SDS buffer so a probe homogenizer can be used to shear DNAs in that case). After sonication, samples were centrifuged at 14,000 rpm for 15 min at 4°C to remove any remaining insoluble material. Concentration of the proteins were measured using Qubit fluorometer (ThermoFisher Scientific). 50 µg of the samples were taken for trypsin digestion. 5 µL of 50 mM ABC containing 5 µg/µL DTT was added, and the sample was incubated at 650C for 15 min followed by addition of 5 µL of 50 mM ABC containing 15 µg/µL iodoacetamide (incubated at RT for 15 min in the dark). Samples were then acidified by adding 12% phosphoric acid (1:10 v/v acid to sample). For every 25 µL of samples, 165 µL of TEAB (Triethylammonium bicarbonate, 1M)/MeOH (10:90 v/v) was added and then loaded to S-trap for further washes. Samples was centrifuged at 4000g for 3 min (4C) to remove supernatant. 150 µL of TEAB (Triethylamonium bicarbonate, 1M)/MeOH (10:90 v/v) was added to the trap as wash solution and the trap was washed 3-6 times depends on the initial loading volume. After the final wash, sequencing grade trypsin dissolved in 50 mM TEAB was added and digested O/N at 370C. The following day, peptides were eluted from the trap by adding 40 µL of 50mM TEAB, 0.1% FA and 0.1% FA in Acetonitrile (50:50), sequentially. The sample was pooled together and dried in a vacufuge and resuspended in 20 µL of 50 mM acetic acid. Peptide concentration was determined by nanodrop (A280 nM).", "projectTags" : [ ], "keywords" : [ "Β2 adrenergic receptors", "Neutrophils", "Infarction" ], "doi" : "10.6019/PXD074944", "submissionType" : "COMPLETE", "publicationDate" : "2026-03-20", "updatedDate" : "2026-02-26", "submissionDate" : "2026-02-26", "downloadCount" : 1, "avgDownloadsPerFile" : 0.032258064, "botCount" : 0, "hubCount" : 0, "organicCount" : 1, "percentile" : 0, "yearlyDownloads" : [ { "year" : "2026", "count" : 1 } ], "submitters" : [ "Felicia Antohe" ], "labPIs" : [ "Felicia Antohe" ], "affiliations" : [ "Institute of Cellular Biology and Pathology" ], "instruments" : [ "Orbitrap Fusion" ], "softwares" : [ ], "quantificationMethods" : [ "Spectrum counting" ], "sampleAttributes" : [ "myocardial ischemia", "Mus musculus (Mouse)", "neutrophil" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Neutrophil" ], "diseases" : [ "Myocardial ischemia" ], "references" : [ ], "experimentTypes" : [ "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "060723_70581_CTRL2_1ug.raw", "70581_CTRL1_1ug.MGF", "060723_70581_CTRL3_1ug.raw", "70581_CTRL3_1ug.MGF", "70581_CTRL4_1ug.MGF", "70581_NE2_1ug.mzid", "060723_70581_CTRL1_1ug.raw", "70581_CTRL3_1ug.mzid", "060723_70581_NE3_1ug.raw", "70581_CTRL1_1ug.mzid", "70581_CTRL4_1ug.mzid", "checksum.txt", "060723_70581_NE2_1ug.raw", "70581_P_NE1_1ug.mzid", "060723_70581_P_NE3_1ug.raw", "060723_70581_NE1_1ug.raw", "70581_P_NE3_1ug.mzid", "060723_70581_CTRL4_1ug.raw", "70581_NE1_1ug.mzid", "70581_CTRL2_1ug.mzid", "70581_P_NE2_1ug.MGF", "060723_70581_P_NE2_1ug.raw", "70581_P_NE1_1ug.MGF", "70581_NE3_1ug.mzid", "70581_NE1_1ug.MGF", "060723_70581_P_NE1_1ug.raw", "70581_NE2_1ug.MGF", "70581_NE3_1ug.MGF", "70581_P_NE2_1ug.mzid", "70581_CTRL2_1ug.MGF", "70581_P_NE3_1ug.MGF" ], "highlights" : { } }, { "accession" : "PXD074961", "title" : "4D-DIA proteomic analysis of SMT-induced cytotoxicity in B16F10 mouse melanoma cells", "projectDescription" : "This dataset contains a 4D-DIA proteomic analysis of B16F10 mouse melanoma cells treated with SMT (SM@CMC-KP101). B16F10 cells were divided into two groups: a PBS control group and an SMT treatment group. After drug exposure, cellular proteins were extracted, digested and analyzed by LC-MS/MS using a Q Exactive HF mass spectrometer. The aim of this study is to identify differentially expressed proteins and perturbed pathways associated with the cytotoxic and anti-tumor effects of SMT on melanoma cells, providing insights into its molecular mechanisms of tumor cell killing.", "dataProcessingProtocol" : "Proteomic analyses were performed by Shanghai OE Biotech Co., Ltd. using a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) equipped with a Nanospray Flex source. Peptides were separated on a C18 analytical column (25 cm × 75 µm) at a flow rate of 300 nL/min. The LC gradient was: 0–40 min, 5–28% solvent B; 40–60 min, 28–42% solvent B; 60–65 min, 42–90% solvent B; 65–75 min, 90% solvent B. The MS acquisition range was 350–1650 m/z, with a resolution of 70,000 for full MS scans and 35,000 for MS/MS scans. The ten most intense precursor ions were selected for higher-energy collisional dissociation (HCD) with a normalized collision energy of 30. A 4D-DIA data acquisition strategy was employed. Raw MS data were processed and searched using MaxQuant (version X.X; please specify the exact version) with default parameters for DIA analysis. Spectra were searched against the UniProt Mus musculus reference proteome database (downloaded on [date]). Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine and N-terminal acetylation were set as variable modifications. Trypsin/P was specified as the digestion enzyme with up to two missed cleavages allowed. Peptide-spectrum matches and protein identifications were filtered at a false discovery rate (FDR) of 1% at both peptide and protein levels. Quantitative analysis was based on DIA signal intensities as implemented in MaxQuant, and downstream statistical analyses were performed using standard workflows.", "sampleProcessingProtocol" : "B16F10 mouse melanoma cells were seeded onto six-well plates at 2.0 × 10^6 cells/well and cultured for 24 h. Cells were then treated with PBS (control group) or SM@CMC-KP101 (SMT treatment group) at a final concentration of 4.0 × 10^7 CFU/mL and incubated for 12 h at 37 °C. After incubation, cells were collected and immediately frozen at −80 °C until proteomic analysis. Frozen cell pellets (approximately 100 mg) were quickly ground into a fine powder in liquid nitrogen and lysed in 300 µL lysis buffer supplemented with 1 mM PMSF. Samples were further lysed by sonication (1 s on/1 s off at 80 W for 2 min) on ice and centrifuged at 12,000 rpm for 10 min at 4 °C to remove insoluble material; a second centrifugation step was performed to further clarify the supernatant. Protein concentration was determined using a BCA assay, and protein aliquots were stored at −80 °C. For SDS-PAGE quality control, 10 µg protein from each sample was separated on 12% SDS-PAGE gels, stained with Coomassie Brilliant Blue, washed for 15 min and scanned using an automatic digital gel image analysis system (Tanon 1600). Based on the measured protein concentrations, 50 µg protein from each sample was adjusted to the same concentration. DTT was added to a final concentration of 5 mM and samples were incubated at 55 °C for 30–60 min, followed by cooling to room temperature. Iodoacetamide was then added to a final concentration of 10 mM and samples were incubated in the dark for 15–30 min. Proteins were precipitated with six volumes of pre-cooled acetone at −20 °C overnight and centrifuged at 8,000 × g for 10 min at 4 °C to collect the pellets. Pellets were resuspended in 100 µL of 50 mM NH4HCO3, and proteins were digested with trypsin at an enzyme-to-substrate ratio of 1:50 (w/w) at 37 °C for 12 h. Digestion was stopped by adjusting the pH to 3 with phosphoric acid, and peptides were desalted using SOLA™ SPE cartridges. After vacuum drying, peptides were resuspended in buffer containing iRT peptides (1:10) prior to LC-MS/MS analysis.", "projectTags" : [ ], "keywords" : [ "B16f10", "Quantitative proteomics", "Sm@cmc-kp101", "Proteomics", "Mouse melanoma", "Lc-ms/ms", "Smt", "4d-dia" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-04-02", "updatedDate" : "2026-02-26", "submissionDate" : "2026-02-26", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "lihao Ji" ], "labPIs" : [ "Wei wei" ], "affiliations" : [ "State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Life Sciences, Nanjing University, Nanjing 210093, P. R. China." ], "instruments" : [ "Orbitrap Astral", "Q Exactive HF" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "cell culture", "melanocyte", "Mus musculus (Mouse)" ], "organisms" : [ "Mus musculus (mouse)" ], "organismsPart" : [ "Cell culture", "Melanocyte" ], "diseases" : [ ], "references" : [ "null--pubMed:0--doi: 10.1038/S41467-026-70949-4" ], "experimentTypes" : [ "Data-dependent acquisition" ], "sdrf" : "", "projectFileNames" : [ "10290-2.raw", "checksum.txt", "10290-1.raw", "proteomics_results.zip", "10290-3.raw", "10290-6.raw", "10290-5.raw", "10290-4.raw" ], "highlights" : { } }, { "accession" : "PXD074932", "title" : "Beyond envenomation: functional dissection of Podalia orsilochus venom uncovers a procoagulant protein and additional toxic activities", "projectDescription" : "Envenomation by urticating caterpillars of the family Megalopygidae is an underestimated public health issue in South America, with Podalia orsilochus being the main species implicated in cases reported in northeastern Argentina. This study aimed to characterize the toxicological properties of its venom, focusing on hematological and hemostatic effects. Venom extracts were analyzed using electrophoresis, zymography, hemolysis and coagulation assays, and chromatographic fractionation followed by immunodetection. Electrophoretic profiles revealed predominant proteins in the 10–37 kDa range, with several gelatinolytic bands detected by zymography. The venom exhibited thrombin-like and dose-dependent procoagulant activity, although weaker than that of Lonomia obliqua. A mild, direct hemolytic effect on human erythrocytes was observed, associated with degradation of Band 3, a major membrane protein. A 33-kDa protein was purified and shown to induce procoagulant activity while cross-reacting with antibodies against Losac, a Factor X-activating protein from L. obliqua venom, indicating the presence of Losac-like toxins in P. orsilochus. In conclusion, P. orsilochus venom contains components with proteolytic, hemolytic, and coagulotoxic activities, including a Losac-like protein, expanding current knowledge of lepidopteran venoms and their potential impact on human health.", "dataProcessingProtocol" : "Raw data were processed using Proteome Discoverer software (version 2.2; Thermo Scientific) and searched against a Lepidoptera protein sequence database with trypsin specificity and a maximum of one missed cleavage per peptide. Searches were performed with a precursor mass tolerance of 10 ppm and product ion tolerance of 0.05 Da. Static modifications included carbamidomethylation of cysteine, and dynamic modifications included methionine oxidation and N-terminal acetylation. Protein hits were filtered for high-confidence peptide matches with a maximum protein and peptide false discovery rate of 1%, calculated using a reverse database strategy. Additional searches were conducted against databases containing venom-related sequences from various caterpillar species, including Megalopygidae, Lonomia obliqua prothrombin activator protein, and proteins reported in UniProt for Podalia orsilochus and L. obliqua. Raw mass spectrometry data files were processed using standard proteomics workflows. Database searches were performed using appropriate search engines against a protein database relevant to the studied organism, including common contaminant proteins. Trypsin was specified as the proteolytic enzyme, allowing for a limited number of missed cleavages. Mass tolerances were set according to instrument specifications. Variable modifications included common post-translational modifications, and false discovery rate (FDR) was controlled at 1% at the peptide and protein levels. The resulting identifications were used for downstream qualitative analysis.", "sampleProcessingProtocol" : "Proteomic analyses were performed at the Proteomics Core Facility CEQUIBIEM (University of Buenos Aires/CONICET) using a protein fraction of 33 kDa obtained by RP-HPLC and excised from an SDS-PAGE gel band stained with Coomassie G-250. Briefly, 50 µg of protein was treated with 8 M urea for 15 min at 80 °C, 100 mM dithiothreitol (DTT) for 30 min at 60 °C, and then 150 mM iodoacetamide (IAA) for 30 min at room temperature to disrupt tertiary structures and reduce/alkylate disulfide bonds. Samples were diluted in 50 mM Tris-HCl buffer (pH 7.5) and digested with sequencing-grade trypsin (20 ng/µL; Promega V5111) in 50 mM ammonium bicarbonate. Digestion was carried out overnight at 37 °C and stopped with 5% formic acid (FA). Peptides were desalted prior to LC-MS/MS analysis using C18 zip tips (Merck Millipore), according to the manufacturer’s instructions. The digests were analyzed by nanoLC-MS/MS using a Thermo Scientific Q Exactive mass spectrometer coupled to an EASY-nLC 1000 nanoHPLC system (Thermo Scientific). Approximately 1 µg of peptides was loaded onto a reverse-phase column (C18, 2 µm, 100 Å, 50 µm × 150 mm; Easy-Spray Column PepMap RSLC, P/N ES801) and eluted over 120 min. The flow rate was 300 nL/min with a gradient from 7% solvent B (5 min) to 35% (120 min). Solvent A was 0.1% FA in water, and solvent B was 0.1% FA in acetonitrile. Injection volume was 2 µL. The MS system included a high-energy collision dissociation (HCD) cell for fragmentation and an Orbitrap analyzer. Electrospray ionization was performed at 3.5 kV using an EASY-Spray source. XCalibur 3.0.63 (Thermo Scientific) was used for data acquisition and instrument control, enabling peptide identification during chromatographic separation. Full-scan MS spectra were acquired in the Orbitrap analyzer over a mass range of 400–1800 m/z at a resolution of 70,000 at 400 m/z. The 12 most intense ions per cycle were sequentially isolated, fragmented by HCD, and measured in the Orbitrap. Peptides with a charge of +1 or unassigned charge state were excluded from MS2 fragmentation.", "projectTags" : [ ], "keywords" : [ "Hemolysis", "Megalopygidae", "Losac", "Proteolytic enzymes", "Procoagulant activity", "Toxins" ], "doi" : "", "submissionType" : "PARTIAL", "publicationDate" : "2026-03-29", "updatedDate" : "2026-02-26", "submissionDate" : "2026-02-26", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Maria Pia Valacco" ], "labPIs" : [ "Maria Elisa Paichoto" ], "affiliations" : [ "INSTITUTO DE BIOLOGIA SUBTROPICAL - NODO PUERTO IGUAZU - CENTRO CIENTIFICO TECNOLOGICO CONICET - NORDESTE - CONSEJO NACIONAL DE INVESTIGACIONES CIENTIFICAS Y TECNICAS -" ], "instruments" : [ "Q Exactive" ], "softwares" : [ ], "quantificationMethods" : [ ], "sampleAttributes" : [ "Megalopygidae sp. BOLD:AAA3544" ], "organisms" : [ "Megalopygidae sp. bold:aaa3544" ], "organismsPart" : [ ], "diseases" : [ ], "references" : [ "Gritti MA, Martínez ME, Gonzalez KY, Lobo López K, Teibler GP, Santoro ML, Peichoto ME. Beyond envenomation: functional dissection of Podalia orsilochus venom uncovers a procoagulant protein and additional toxic activities. Toxicon. 2026 276:109086--pubMed:41881159--doi: 10.1016/j.toxicon.2026.109086" ], "experimentTypes" : [ "Data-dependent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "Contaminantes.fasta", "checksum.txt", "MEP32.raw", "MEP32a.msf", "MEP32b.msf" ], "highlights" : { } }, { "accession" : "PXD074943", "title" : "Mulit-omics analysis of keratinocytes reveals dermokine as regulator keratinocyte differentiation and adhesion - p120 perturbed proteome", "projectDescription" : "Impaired adhesion and differentiation of keratinocytes is a hallmark of several skin diseases, but only some of the factors that regulate these processes have been identified. Here, we studied the role of isoform-rich dermokine – a wound- and tumor-regulated protein – in keratinocytes using a combination of multi-omics and functional approaches. CRISPR/Cas9-induced knockout of dermokine isoforms in human keratinocytes inhibited differentiation of these cells in three-dimensional organotypic skin cultures, which was confirmed by quantitative proteomics. In two-dimensional monocultures, dermokine deficiency affected the proteome and phosphoproteome as revealed by mass spectrometry. We found reduced abundance of differentiation-specific proteins and increased phosphorylation of cell adhesion protein p120 (catenin-δ1). The adhesive strength of dermokine knockout keratinocytes was impaired, which was rescued by p120 knock-down or ROCK inhibition. Finally, we verified the correlation between decreased dermokine expression and increased p120 phosphorylation in human non-healing wounds. These results identify dermokine as regulator of keratinocyte adhesion and differentiation, involving at least in part its effect on p120 phosphorylation and ROCK. Our data point to a function of dermokine in the pathogenesis of chronic wounds.", "dataProcessingProtocol" : "Raw files were analysed using Spectronaut™ (Biognosys) directDIA+ (deep) default settings, spectra were matched against the homo sapiens reference FASTA database (UP000005640; downloaded from Uniprot on 27th of December 2022). Default settings were used without imputation. Dynamic modifications were set as Oxidation (M), Glutamine to pyro-Glutamine and Acetyl on protein N-termini. Cysteine carbamidomethyl was set as a static modification. Protein quantitation was done on the MS2 level, data filtering set to default Q-values (1 % FDR). Data were processed using R (version 4.1.1). Gene ontology analysis was performed using Metascape and each exported term a module-dependent p value was calculated using Stouffer’s Z method. Resulting p values were adjusted for multiple testing using Benjamini Hochberg corrections.", "sampleProcessingProtocol" : "Lysis buffer (4M guanidine hydrochloride (GuHCl) and 250 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) (pH 7.8)) was added to 3D organotypic skin cultures, which were then homogenized using the steel beads (QIAGEN) and TissueLyser II (QIAGEN). The lysate was processed in the TissueLayser twice for 1 min each at varying frequencies (3 Hz to 30 Hz), beads were removed using a magnetic rack and the suspension was heated for 5 min at 95 °C. Following this, the samples underwent five cycles of sonication (30 Hertz for 30 seconds each) and cooling using the TissueLyser ii (QIAGEN). After centrifugation (13,000 x g for 15 minutes at 4 °C), the protein content was determined using both the Nanodrop (Thermo Fisher Scientific) and Quickstart Bradford Protein assay (Bio-Rad; Cat#5000001). The samples were reduced and alkylated on aliquots of 50 µg protein using 5 mM tris(2-carboxyethyl)phosphin (TCEP) and 20 mM 2-chloroacetamide (CAA). The samples were diluted with 50 mM HEPES buffer (pH 7.8) and incubated with Lys-C (FUJIFILM) for 4 h, 37 °C followed by trypsin (Promega) for 16 h at 37 °C. The enzyme activity was blocked by 1% tri-fluoroacetic acid (TFA) and samples were desalted using Solaµ plates (Thermo Fisher Scientific), according to manufacturer’s instructions. Peptides were eluted using a 58-minute gradient on the EvoSep One instrument and analysed using the Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific). Spray voltage was set to 2.3 kV, funnel RF level at 40, and heated capillary at 240 °C. Full MS spectra were collected at a resolution of 120000, with an AGC target of 300% or maximum injection time set to ‘custom’ and a scan range of 400–1200 m/z. Operating in DIA mode, we set the MS2 resolution to 60000, scan range 200 – 1200 m/z, maximum injection time to 118 ms and the HCD value to 28 %. We set the CV value to a single -50 V. The isolation window was set to 8 m/z with a 1 m/z overlap and window placement optimization on. The maximum number of scan event was set to 99, loop to 3 s and loop count to 25.", "projectTags" : [ ], "keywords" : [ "" ], "doi" : "", "otherOmicsLinks" : [ "px:PXD050151" ], "submissionType" : "PARTIAL", "publicationDate" : "2026-04-09", "updatedDate" : "2026-02-26", "submissionDate" : "2026-02-26", "downloadCount" : 0, "avgDownloadsPerFile" : 0.0, "percentile" : 0, "submitters" : [ "Vahap Canbay" ], "labPIs" : [ "Chiara Francavilla" ], "affiliations" : [ "Department for Bioengineering and Biomedicine, Technical University of Denmark, 2800, Copenhagen, Denmark." ], "instruments" : [ "Orbitrap Eclipse" ], "softwares" : [ "Spectronaut" ], "quantificationMethods" : [ "MS1 intensity based label-free quantification method" ], "sampleAttributes" : [ "Wounds and Injuries", "keratinocyte", "Homo sapiens (Human)", "skin", "epithelial cell" ], "organisms" : [ "Homo sapiens (human)" ], "organismsPart" : [ "Keratinocyte", "Epithelial cell", "Skin" ], "diseases" : [ "Wounds and injuries" ], "references" : [ ], "experimentTypes" : [ "Data-independent acquisition", "Bottom-up proteomics" ], "sdrf" : "", "projectFileNames" : [ "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_WT_Lipofectamine_3.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_B11_p120siRNA_1.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_CS9_p120siRNA_2.raw", "20260218_173833_p120_siRNA_20250904.sne", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_B11_scrambled_3.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_WT_p120siRNA_3.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_CS9_scrambled_3.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_WT_Lipofectamine_1.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_B11_p120siRNA_3.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_B11_scrambled_1.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_CS9_scrambled_1.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_WT_scrambled_2.raw", "checksum.txt", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_CS9_p120siRNA_3.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_B11_p120siRNA_2.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_WT_Lipofectamine_2.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_WT_p120siRNA_1.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_CS9_p120siRNA_1.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_WT_p120siRNA_2.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_B11_scrambled_2.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_CS9_scrambled_2.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_WT_scrambled_1.raw", "20250904_HB_MEKONG_VC_FAIMS_1CV_Aurora15_20SPD_0000_DIA_400ng_WT_scrambled_3.raw" ], "highlights" : { } } ]