PXD000225

PXD000225 is an original dataset announced via ProteomeXchange.

Title	Phosphoproteomics study on protein kinase C delta induced cell death
Description	The proteolytic activation of protein kinase C delta generates a catalytic fragment called PKC delta-CF, which induces cell death. However, the mechanisms underlying PKC delta-CF-mediated cell death are largely unknown. On the basis of an engineering leukemic cell line with inducible expression of PKC delta-CF, here we employ SILAC-based quantitative phosphoproteomics to systematically and dynamically investigate the overall phosphorylation events during cell death triggered by PKC delta-CF expression. Totally, 3000 phosphorylation sites were analyzed. We combined SILAC for quantitation, strong-cation exchange chromatography and titanium dioxide chromatography for phosphopeptide enrichment, and high-accuracy mulistage MS. All MS/MS ion spectra were analyzed using Sequest version v.27, rev. 1146 (Thermo Fisher Scientific, San Jose, CA) which were incorporated into the Sorcerer engine version 4.0.4 build (Sage-N Research). Sequest were set up to search the ipi.HUMAN.v3.65 database (86379 entries) (ftp.ebi.ac.uk/pub/data bases/IPI/current) with its target-decoy database as a reversed complement assuming the semi-enzyme tryptic digestion allowed for three missed tryptic cleavages with full mass from 600 to 4600. The precursor ion tolerance was set to ±10 p.p.m and MS2 ions tolerance was set to 1 Da. Searches parameters for non-phosphopeptides were allowed for a static modification of Carbamidomethyl cysteine (C, +57.021465) and dynamic modifications of oxidized methionine (M, +15.99492) on, SILAC-labeled lysine (K, 6C2N, +8.014199) on and SILAC-labeled arginine (R, 6C4N, +10.008269) with up to 4 total dynamic modifications and up to 3 of any particular dynamic modification. In addition, searches for phosphopeptides were allowed for dynamic modifications of phosphorylation serine (S), threonine (T), and tyrosine (Y) (+79.966331) with up to 6 total dynamic modifications and up to 4 of any particular dynamic modification. The ASCORE algorithm was selected to assess the possibility of phosphorylation sites. DATSelect 2.0.39 was used to filter and group the data files derived from SORCERER-SEQUEST. The filter thresholds were set to XCorr 2+, 3+, 4+: > 2.5, > 3.0, > 3.5 and DeltaCN > 0.08. For protein identification, two peptides were required with estimated false-discovery rates (FDR) < 1%. For phosphopeptide identification, only unique peptide was required with FDR <2%, whereas DeltaCN should be adjusted to >0.125 if the FDR for phosphopeptide assignments was >2%. Furthermore, additional filtering was used to remove those indubitably false phosphopeptides, including sequences that contained both heavy and light isotopic variants of arginine and lysine. Finally, the FDR fall within 1%.
HostingRepository	PRIDE
AnnounceDate	2013-08-08
AnnouncementXML	Submission_2013-08-08_08:17:19.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Li Xia
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue; iodoacetamide derivatized residue; monohydroxylated residue; 6x(13)C: 2x(15)N labeled L-lysine; 6x(13)C: 4x(15)N labeled L-arginine
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2013-04-25 01:42:42	ID requested
1	2013-07-26 10:57:37	announced
1	2014-07-25 01:05:11	announced
⏵ 2	2013-08-08 08:17:19	announced	Add reference

Xia L, Wang TD, Shen SM, Zhao M, Sun H, He Y, Xie L, Wu ZX, Han SF, Wang LS, Chen GQ, -induced cell death. J Proteome Res, 12(10):4280-301(2013) [pubmed]

submitter keyword: pkcdelta phosphoproteomics apoptosis

Li Xia
contact affiliation	Pathophysiology
contact email	laurent_xia@yahoo.com.cn

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