Project PXD003928

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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.


Adán Alpízar, CNB
Jose Maria Carazo, Estructura de Macromoléculas (CNB-CSIC) ( lab head )

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Shotgun proteomics

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    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


Showing 1 - 8 of 8 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 63231 CM40_Pool 7842 88942 57873 51928 27013
2 63233 CM50_Pool 3472 27317 16611 12606 8511
3 63232 CM70_Pool 3156 29641 15855 15633 9343
4 63235 CM40_Pool 4274 47257 27220 40784 17054
5 63234 CM70_Pool 5122 63351 34847 59672 23189
6 63237 CM40_Pool 3047 35915 16657 22461 13114
7 63236 CM50_Pool 786 6469 2647 4692 2887
8 63238 CM50_Pool 9382 118754 79759 84684 38157