PXD000242

PXD000242 is an original dataset announced via ProteomeXchange.

Title	Iloprost (2/5 nM) stimulated Platelets
Description	TiO2 enriched phosphopeptides of Iloprost stimulated platelets were digested with trypsin and labeled with iTRAQ. Samples were analysed by nano LC-MS/MS on an LTQ OrbitrapVelos (Thermo Scientific) coupled to an Ultimate 3000 Liquid Chromatography system (Dionex). Peptides were concentrated on a self-packed 100 µm ID reversed-phase (RP) trapping column (Kinetex C18, 2 cm length, 2.6 µm particle size, 100 Å pore width; Phenomenex) in 0.1% TFA followed by separation on a self-packed 75 μm ID RP column (Kinetex C18, 30 cm length, 2.6 µm particle size, 100 Å pore width; Phenomenex) using a binary gradient (solvent A: 0.1% FA and solvent B: 0.1% FA, 84% ACN) ranging from 3-45 % solvent B in 245 min at a flow rate of 230 nl/min. To minimize memory and carry-over effects from previous samples, an LC-wash program with high organic content was used prior to sample injection (Burkhart et al 2011). Survey scans were acquired in the Orbitrap with a resolution of 30,000 using the polysiloxane m/z 371.101236 as lock mass5 and MS/MS scans of the five most intense signals were acquired in the Orbitrap using HCD fragmentation (activation 0.1 ms, normalized collision energy of 50, isolation width of 1.5 m/z) with a resolution of 7,500. To compensate for elevated peptide charge states due to iTRAQ labels, a reaction tube containing 5 % ammonium water was placed in front of the ion source as described by Thingholm et. al. Database search and data processing: Raw data were processed with Proteome Discoverer 1.3 (Thermo Scientific) using Mascot (2.4) and Sequest for database search against the human Uniprot database (November, 4th 2010; 20,260 sequences) using the following search settings for both algorithms: i) trypsin with a maximum of two missed cleavages, (ii) carbamidomethylation of Cys (+57.0214 Da), peptide N-terminus iTRAQ, Lys iTRAQ (+144.102 Da) as fixed modifications and (iii) oxidation of Met (+15.9949 Da), Tyr iTRAQ (+144.102 Da) as well as phosphorylation of Ser/Thr/Tyr (+79.9663 Da) as variable modifications; (iv) MS and MS/MS tolerances of 10 ppm and 0.02 Da, respectively. A false discovery rate (FDR) of <1% was applied using the Peptide Validator node. Additionally, phosphorylation site localizations were validated by the phosphoRS node. Only phosphopeptide hits with <1% FDR of all validated peptides were considered for data interpretation and site assignment probabilities of >90 % (phosphoRS) were defined as confident. Data interpretation: To compensate for systematic variations or technical errors for each sample set, reporter areas were normalized based on modellized normal distribution with a +- 34% quantil. Therefore median iTRAQ ratios were calculated considering all unique <1% FDR peptides and normalization factors against the control sample were determined. For each peptide, all MS/MS spectra of one biological replicate were grouped in regard to modified site in protein and phosphoRS site localization (peptide groups) to determine the respective median ratios. Finally, for each peptide group, median values were calculated considering all three biological replicates. Peptides with ratios below 0.5 or above 2.0 were considered as regulated. Regulated phosphopeptides were searched against GPS 2.1 for kinase consensus site prediction. Values of high confidence (<2 % error) for PKA were taken into account.
HostingRepository	PRIDE
AnnounceDate	2014-07-25
AnnouncementXML	Submission_2014-07-25_01:08:45.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Florian Beck
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; iTRAQ4plex-116 reporter+balance reagent acylated residue; iodoacetamide derivatized residue; phosphorylated residue
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2013-05-06 14:05:12	ID requested
1	2013-12-02 07:42:10	announced
1	2013-12-13 06:59:43	announced
⏵ 2	2014-07-25 01:08:46	announced	Updated project metadata.

Dataset with its publication pending

submitter keyword: iTRAQ, Iloprost, Phosphopeptides, Platelets

Florian Beck
contact affiliation	Leibniz Institut fuer Analytische Wissenschaften ISAS e.V.
contact email	florian.beck@isas.de
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2013/12/PXD000242

PRIDE project URI

• PRIDE
  1. PXD000242
     1. Label: PRIDE project
     2. Name: Iloprost (2/5 nM) stimulated Platelets