PXD000577

PXD000577 is an original dataset announced via ProteomeXchange.

Title	PhosFox: a software tool for peptide-level processing of LC-MS/MS-based phosphoproteomic data
Description	Human keratinocytes, HaCaT cells were transfected with the dsRNA-analogue polyinosinic-polycytidylic acid (poly I:C) (Sigma-Aldrich) using Lipofectamine 2000 reagent (Invitrogen) for intracellular delivery. The cells were transfected with 7 ug/ml poly I:C for 1 h or left untreated. The cells were collected in PBS with a cell scraper and washed once with PBS before they were lysed with HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, pH 7.4) including protease and phosphatase inhibitor cocktails (Sigma-Aldrich). The cell lysates were centrifuged 2,264 x g for 15 min at 4°C, divided into smaller fractions and centrifuged again 11,686 x g for 15 min at 4°C . The supernatant was collected and the protein content was measured with Bio-Rad DC protein assay (Bio-Rad). For the samples, 8 mg of protein was used. The proteins were precipitated with 10 % TCA/acetone at -20°C overnight. Samples were centrifuged 10,733 x g for 20 min at 4°C and the supernatant was removed. The precipitation was washed once with acetone and dissolved in urea buffer (8 M urea, 400 mM NH4CO3, 20 mM DL-dithiotheitol, 1 mM EDTA, pH 8.5). The proteins were reduced, alkylated, and enzymatically digested in-solution with lysyl endopeptidase (Wako Chemicals) and trypsin (Promega). The peptides were fractionated by strong cation exchange chromatography. Phosphopeptide enrichment was performed with immobilized-metal affinity chromatography (IMAC) using PHOS-Select Iron Affinity Gel (Sigma Aldrich). The enriched phosphopeptides were analyzed by nanoLC-MS/MS using an Ultimate 3000 nano-LC (Dionex) coupled to a QSTAR Elite hybrid quadrupole TOF-MS (Applied Biosystems/MDS Sciex) with nano-ESI ionization.23,24 The LC-MS/MS data were submitted through the ProteinPilot 4.0 interface (Applied Biosystems/MDS Sciex) to an in-house Mascot database search engine version 4.0 (Matrix Science), and to the ProteinPilot algorithm Paragon. The data were searched against the human canonical sequences in the Swiss-Prot database (decoy version 08132012 for the Mascot searches and version 04012013 with 539,829 sequences for the Paragon searches). The Mascot search criteria were: fixed modifications: carbamidomethyl of cysteine, variable modifications: oxidation (M), phospho (ST), phospho (Y), acetylation (K), 1 missed cleavage allowed, enzyme: trypsin. The Paragon search criteria were: modifications: cystein alkylation with iodoacetamide, ID focus: biological modifications, phosphorylation emphasis, identification threshold: 95 % confidence (Unused ProtScore (Conf) > 1.3), Thorough search (ID), enzyme: trypsin.
HostingRepository	PRIDE
AnnounceDate	2014-07-25
AnnouncementXML	Submission_2014-08-08_06:05:50.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Tuula Nyman
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	acetylated residue; iodoacetamide derivatized residue; phosphorylated residue
Instrument	QSTAR

Revision	Datetime	Status	ChangeLog Entry
0	2013-11-12 02:02:45	ID requested
1	2014-06-30 01:07:31	announced
2	2014-07-25 01:46:30	announced	Updated project metadata.
⏵ 3	2014-08-08 06:05:50	announced	Updated project metadata.

S, ö, derholm S, Hintsanen P, Ö, hman T, Aittokallio T, Nyman TA, PhosFox: a bioinformatics tool for peptide-level processing of LC-MS/MS-based phosphoproteomic data. Proteome Sci, 12():36(2014) [pubmed]

submitter keyword: human, LC-MS/MS, phosphorylation, peptide

Tuula Nyman
contact affiliation	Institute of Biotechnology
contact email	tuula.nyman@helsinki.fi
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/2014/06/PXD000577

PRIDE project URI

• PRIDE
  1. PXD000577
     1. Label: PRIDE project
     2. Name: PhosFox: a software tool for peptide-level processing of LC-MS/MS-based phosphoproteomic data