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Mouse bone marrow derived neutrophils, LC-MSMS
Analysis of cell membrane-enriched proteins of mouse polymorphonuclear neutrophils. Further related data are present in project PXD002244.
Sample Processing Protocol
Bone marrow derived neutrophils, isolated from NOR/Lt mice, were suspended in Tris-EDTA buffer (25mM Tris, 1mM EDTA, pH:7.5) containing 7% protease inhibitor cocktail (Sigma-Aldrich) and sonicated by two 15 second-cycles (VibraCell, Bioblock). Unbroken cells, nuclei, and cell debris were removed by centrifugation at 3,000g for 10min. Microsomes were harvested from supernatant by 100,000g ultracentrifugation at 4°C for 1h (Beckman SW41 rotor) and the membrane-enriched pellet was lysed by a 30min incubation at 4°C in RIPA buffer (Sigma-Aldrich) containing 0.5% SDS and 1% protease inhibitor cocktail. Protein lysate was obtained after a 20,000g centrifugation at 4°C for 15min. Membrane protein samples were run into SDS polyacrylamide gel comprising a 1cm-high stacking 4% w/v polyacrylamide matrix on top of a 20% w/v polyacrylamide matrix, at 80V until protein samples concentrate at the 4-20% w/v gel interface. After staining with Coomassie blue gel fragments were excised and processed separately. After destaining and washing in a 50:50 mixture of acetonitrile (CH3CN) and 50 mM ammonium bicarbonate buffer (NH4HCO3), the proteins were reduced with 10 mM dithiothreitrol (DTT, Sigma-Aldrich) for 1h at 57°C and free cysteine residues were alkylated with 55 mM iodoacetamide (Sigma-Aldrich) for 45min at room temperature. After further washings of gel slices, proteins were next digested with 12 ng/µl trypsin (Promega) in 25 mM NH4HCO3 buffer containing 0,01% ProteaseMAXTM surfactant (Promega) for 3h30 at 37°C. Once the reaction stopped with 1% formic acid, peptide-containing supernatants were sonicated, vortexed and concentrated by vacuum centrifugation.
Data Processing Protocol
The peptides resulting from in-gel trypsin hydrolysis of samples were analyzed by mass spectrometry (MS) using a LTQ-Orbitrap VELOS mass spectrometer (Thermo-Fisher) coupled to a nanoscale liquid-chromatography (LC) system (U3000 RSLC system, Thermo-Fisher). Chromatographic separation was performed on a 50-cm long reverse-phase capillary column (Acclaim Pepmap C18 2-µm 100 A, 75-µm i.d.) using a 1-hour gradient of acetonitrile. Typical survey method was used for the fragmentation of the peptides (MS/MS), in which full MS scans were acquired at 60,000 resolution (FWMH) using the Orbitrap analyzer (on a mass-to-charge ratio (m/z) range of 300 to 2000) while the collision-induced dissociation (CID) spectra (MS/MS) for the eight most intense ions were recorded in the linear LTQ trap. Protein identification was performed with X!Tandem search engine. Mass data were confronted to UniprotKB restricted to “Mus” taxonomy (taxon id 862507; 83192 sequences, release from March 12, 2014). Parameters of the databank search were as follows: mass accuracy for peptides was set at +/- 5ppm, cysteines were modified by iodoacetamide (this modification was introduced during the samples preparation), and possible oxidation of methionines and sulfation of tyrosines were considered.
Haurogné K, Pavlovic M, Rogniaux H, Bach JM, Lieubeau B; Type 1 Diabetes Prone NOD Mice Have Diminished Cxcr1 mRNA Expression in Polymorphonuclear Neutrophils and CD4+ T Lymphocytes., PLoS One, 2015, 10, 7, e0134365, PubMed: 26230114
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||58344||no assay title provided (mzIdentML)||1632||1820||1409||14134||1794||
|2||58345||no assay title provided (mzIdentML)||2251||2632||2177||15281||2622||
|3||58341||no assay title provided (mzIdentML)||3122||2567||2136||16970||2545||
|4||58340||no assay title provided (mzIdentML)||4174||2938||2413||17386||2914||
|5||58343||no assay title provided (mzIdentML)||3979||2625||2166||16443||2604||
|6||58342||no assay title provided (mzIdentML)||3569||2415||2013||15540||2396||