Project PXD002676

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Biological Dataset
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Summary

Title

Proteomic and bioinformatic analysis of the nuclear intrinsically disordered proteome

Description

Intrinsically disordered proteins (IDPs) are biologically active molecules which are involved in many cellular functions although they do not possess a defined three-dimensional structure. They are mostly signalling and regulatory proteins. This study is the first large-scale proteomic analysis of the nuclear IDPs. We experimentally showed that IDPs are overrepresented in the nucleus in comparison to the whole cell. The analysis in terms of molecular function indicated that nuclear intrinsically disordered proteome (IDP-ome) is enriched in proteins involved in transcription regulation and especially in transcription factors.

Sample Processing Protocol

Nuclei from HEK293 cells were isolated in a hypoosmotic buffer containing detergent and further purified by centrifugation in a high-density sucrose solution. Proteins from the obtained nuclei were extracted in a high-salt (350 mM NaCl) lysis buffer. The samples were enriched in IDPs by the heat-treatment method (Galea et al., 2006 and 2009). Three independent experiments using cells from three subsequent passages were conducted. For each replication, samples from nuclear lysate enriched in IDPs (N_IDPs) as well as from not enriched nuclear lysate (N), whole cell lysate (WCL) and whole cell lysate enriched in IDPs (WCL_IDPs) were collected. The received proteins were prepared for a shotgun LC-MS/MS analysis using FASP approach (Wiśniewski et al., 2009). LC-MS/MS measurements were performed using micrOTOF–Q II (Bruker Daltonics) tandem mass spectrometer coupled with nanoHPLC (UltiMate 3000 RSLCnano System, Dionex). Peptides were separated in a 240 min gradient of acetonitrile from 2% to 40% in the presence of 0.05% FA. Mass spectrometry measurement cycle consisted of one MS scan and five subsequent MS/MS scans in data dependent manner.

Data Processing Protocol

The obtained data were internally calibrated and analysed using Data Analysis 4.1 software (Bruker Daltonics). An in-house MASCOT server (v. 2.3.0, Matrix Science) was used for searching against Swissprot_201407 database restricted to Homo sapiens taxonomy (20284 sequences). The searching was done via ProteinScape 3.0 platform (Bruker Daltonics). The parameters were as follows: enzyme – trypsin, number of missed cleavages – 1, fixed modification – carbamidomethylation (C), variable modifications – oxidation (M), deamidation (NQ), peptide mass tolerance – ±20 ppm, fragment mass tolerance – ±0.05 Da and an automatic decoy database search was performed. False Positive Rate (FPR) was set to below 1% at a protein level. After the first LC-MS/MS analysis, a Scheduled Precursor List (SPL) was generated on the basis of search results and the second run was performed with SPL as an exclusion list. At the end, the compilation of the search results from two measurements was done with the use of ProteinScape 3.0 platform. Data submitted to the ProteomeXchange include: Bruker mass spectrometer output files (.baf) and pride xml files generated from Mascot DAT files for each of 24 MS measurements.

Contact

Bozena Skupien-Rabian, 1. Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland 2. Department of Structural Biology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
Sylwia Kedracka-Krok, 1. Department of Structural Biology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland 2. Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland ( lab head )

Submission Date

06/08/2015

Publication Date

21/09/2015

Experiment Type

Shotgun proteomics

Assay count

24

Publication

    Skupien-Rabian B, Jankowska U, Swiderska B, Lukasiewicz S, Ryszawy D, Dziedzicka-Wasylewska M, Kedracka-Krok S. Proteomic and bioinformatic analysis of a nuclear intrinsically disordered proteome. J Proteomics. 2015 Sep 12. pii: S1874-3919(15)30120-2 PubMed: 26376097

Assay

Page 1 2 3
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Showing 1 - 10 of 24 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 56034 Nuclear lysate – replicate #1 1006 7435 3574 24834 6760
2 56029 Nuclear lysate – replicate #1, measurement with an exclusion list 660 2364 1262 17461 2186
3 56035 Nuclear lysate – replicate #2 958 6991 3380 24290 6442
4 56036 Nuclear lysate – replicate #2, measurement with an exclusion list 655 2377 1274 17755 2205
5 56027 Nuclear lysate – replicate #3 986 7355 3506 24168 6799
6 56037 Nuclear lysate – replicate #3, measurement with an exclusion list 656 2480 1281 17576 2301
7 56019 Nuclear lysate enriched in IDPs - replicate #1 680 5136 2116 23983 4842
8 56028 Nuclear lysate enriched in IDPs – replicate #1, measurement with an exclusion list 480 1927 838 18593 1812
9 56038 Nuclear lysate enriched in IDPs – replicate #2 603 4758 1770 23915 4521
10 56025 Nuclear lysate enriched in IDPs – replicate #2, measurement with an exclusion list 386 1901 722 18766 1841