PXD005410

PXD005410 is an original dataset announced via ProteomeXchange.

Title	HiRIEF LC-MS allows for in depth quantitative analysis of the phosphoproteome.
Description	In this project we aimed at improving phosphoproteomics analysis by taking advantage of high resolution isoelectric focusing (HiRIEF) fractionation. We developed a workflow that employs titanium dioxide phospho-enrichment, followed by isobaric labeling with Tandem Mass Tags (TMT) and HiRIEF on a broad pI range (immobilized pH gradient, IPG gel strips employed were 2.5-3.7 and 3-10, Phospho HiRIEF LC-MS). We analyzed HeLa cells untreated (four biological replicates), treated with pervanadate or arrested in mitosis (three biological replicates each). Employing a relatively low amount of material (300 μg of peptides), we identify 22,674 phosphorylation sites, of which 19,036 were localized with high confidence. We demonstrate isoelectric point dependent fractionation of the peptides based on the number of phosphate groups that they carry: 18% of the phospho-peptides identified with the IPG 2.5-3.7 gel strip are multiply phosphorylated peptides and they localize predominantly in the most acidic pI fractions. Identified phosphorylation sites include 1,198 tyrosine phosphorylation sites and 1,491 phospho-sites that were not previously reported in the PhosphoSitePlus database. Total protein quantification performed by standard HiRIEF on the same samples identified 9,185 proteins, of which 4,575 overlap with the proteins identified by Phospho HiRIEF LC-MS. Phosphorylation sites corresponding to these proteins were normalized to total protein abundance, resulting in 18,374 quantified phospho-sites. Kinase association analysis on the quantified phospho-sites resulted in identification of a subset that has putative functions during the mitotic phase and protein-protein interaction network analysis shows a high degree of connectivity of these putatively functional novel phospho-sites.
HostingRepository	PRIDE
AnnounceDate	2017-07-07
AnnouncementXML	Submission_2017-07-10_00:25:20.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Rui Branca
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	TMT6plex-126 reporter+balance reagent acylated residue; phosphorylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2016-11-21 05:57:29	ID requested
1	2017-07-07 08:06:04	announced
⏵ 2	2017-07-10 00:25:22	announced	Updated publication reference for PubMed record(s): 28674419.

Panizza E, Branca RMM, Oliviusson P, Orre LM, Lehti, ö J, Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome. Sci Rep, 7(1):4513(2017) [pubmed]

curator keyword: Technical, Biological

submitter keyword: HiRIEF, fractionation, isoelectric point, LC-MS, phosphorylation, IPG, multiply phosphorylated, phosphoproteomics, proteomics, phospho-peptides, 10-plex, multiplex, TMT, quantitative, quantification, HeLa, cells, cancer, mitosis, mitotic arrest, pervanadate, tyrosine, phospho-sites, kinase association, NetworKIN

Janne Lehtio
contact affiliation	Clinical Proteomics Mass Spectrometry, Science for Life Laboratory, Dept. of Oncology Pathology, Karolinska Institutet
contact email	janne.lehtio@ki.se
lab head
Rui Branca
contact affiliation	Clinical Proteomics Unit, Dep. of Oncology-Pathology
contact email	rui.mamede-branca@ki.se
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/2017/07/PXD005410

PRIDE project URI

• PRIDE
  1. PXD005410
     1. Label: PRIDE project
     2. Name: HiRIEF LC-MS allows for in depth quantitative analysis of the phosphoproteome.