PRIDE Assigned Tags:Biological Dataset
Visualize in PRIDE Inspector
1.Download, uncompress and open PRIDE Inspector
2.Click in the magnifier on the left top corner, paste the project or assay that you would like to open in the search box, and hit search
3.Click in the corresponding "Download" button to download the files and visualize them
Structure and proteomics on centrosome from differentiated and quiescent cells: the thymocyte case
We have applied cryo-tomography and proteomics analyses to centrosomes isolated from young lamb thymus– a convenient source of quiescent and differentiated cells. Those analyses have allow us to better depict the protein organization joining the two centrioles in such a cellular system, in the form of a disperse network of fibers and an amorphous density, both parts linked together. Protein composition of the centrosome have been compared with published data on centrosome extracted from cell culture , and presence of various protein reported implicated in the disengagement and dissociation processes questioned. C-NAP1, cohesin smc1 and furthermore condensin smc4 and ncapd2 have been localized on the amorphous density, however DNA hasn't. Subtomogram averaging processing have been applied to the centriolar wall, both on the proximal microtubule triplets and distal doublets; results have been compared to the already published structures of centriolar wall triplet from distant species basal bodies.
Sample Processing Protocol
For proteomics analysis, each purified fraction (40%, 50% and 70%) was first resolved by gel electrophoresis. Typically, sucrose gradient fractions were centrifuged at 14,000 rpm for 15 min and the pellet was re-suspended in laemmli sample buffer and boiled. Samples were resolved on a 1D-SDS-PAGE 10% stacking gel. Bands of interest from coomasie gels were excised manually, deposited in 96-well plates and processed either manually or automatically in a Proteineer DP (Bruker Daltonics, Bremen, Germany). The reaction was stopped by adding 0.5% TFA for peptide extraction. The tryptic eluted peptides were dried by speed-vacuum centrifugation and kept at -20 ºC until LC-MS/MS analysis. Samples were acquired by LC-MS/MS using a nano HPLC chromatography system (Eksigent Technologies nanoLC Ultra 1D plus) coupled online to a TripleTOF 5600 mass spectrometer (AB SCIEX, Framingham, MA) with a nano-spray ionization source.
Data Processing Protocol
MS and MS/MS data obtained for each sample fraction were processed using Peak View Software v1.1 (AB SCIEX) to generate peaklists in a mascot general file (mgf) format. Database searches were carried out using a licensed in-house Mascot Server v 2.5.0 (Matrix Science, London, UK). Searches were performed against the UniProtKB/SwissProt database (release 09.01.2015) with Ovis aries taxonomy restriction (UKBsp_p9940), containing 23112 protein coding genes and their corresponding reversed entries that was generated in-house using the ProteoWizard program. Search parameters were set as follows: carbamidomethyl cysteine as fixed modification, oxidized methionines, N-term pyroglutamic and acetylation of the peptide amino termini as variable ones. Peptide mass tolerance was set to 25 ppm in MS mode and 0.06 Da in MS/MS mode, and 2 missed cleavages were allowed. Typically, an accuracy of ± 10 ppm was found both for MS and MS/MS spectra. Searches were exported as .dat file from Mascot and uploaded in the Scaffold bioinformatic tool v4.0 (Proteome Software, Inc. Oregon), combining replicate analyses into the same treatment conditions, enabling sample inter-protein grouping and FDR filtering. False Discovery Rates (FDR ≤ 1% at the protein level) for protein identification were applied using the automatic filters from the software. Other additional filters used were: proteins identified with at least two peptides, protein identification 98% greater probability and proteins should have been identified with at least a unique peptide.
Busselez J, Chichón FJ, Rodríguez MJ, Alpízar A, Gharbi SI, Franch M, Melero R, Paradela A, Carrascosa JL, Carazo JM. Cryo-Electron Tomography and Proteomics studies of centrosomes from differentiated quiescent thymocytes. Sci Rep. 2019 9(1):7187 PubMed: 31076588
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|