PXD000599

PXD000599 is an original dataset announced via ProteomeXchange.

Title	Phosphoproteomic analysis of breast epithelial cells with isogenic knockin of PIK3CA mutations
Description	The PIK3CA gene is frequently mutated in human cancers. To study the signaling mechanisms responsible for cell growth and invasion phenotypes induced by mutant PIK3CA molecules, we carried out a SILAC-based quantitative phosphoproteomic analysis of MCF10A, a spontaneously immortalized normal mammary epithelial cell line, and two MCF10A knockin cell lines containing different activating mutations of the PIK3CA gene. MCF10A and PIK3CA mutation knock in cells were propagated in DMEM/F12 SILAC media deficient in both L-lysine and L-arginine and supplemented with light lysine (K) and arginine (R) for light, 2H4-K and 13C6-R for medium state and 13C615N2-K and 13C615N4-R for heavy state labeling. Cell lysates were prepared in urea lysis buffer containing 20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate and 5mM sodium fluoride. The lysates were reduced, alkylated and digested by trypsin. Tryptic peptides were desalted by C18 reverse phase column and followed by strong cation exchange (SCX) fractionation. Fractionated peptides were subjected to TiO2-based phosphopeptide enrichment. LC-MS/MS analysis of enriched phosphopeptides was carried out using a reverse-phase liquid chromatography system interfaced with an LTQ-Orbitrap Velos mass spectrometer. Proteome Discoverer (v 1.3) suite was used for quantitation and database searches. The tandem mass spectrometry data were searched using Mascot (2.2.0) and SEQUEST search algorithms against a Human RefSeq database supplemented with frequently observed contaminants.
HostingRepository	PRIDE
AnnounceDate	2015-07-28
AnnouncementXML	Submission_2015-07-28_09:35:02.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Akhilesh Pandey
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	6x(13)C: 4x(15)N labeled L-arginine; phosphorylated residue; 6x(13)C: 2x(15)N labeled L-lysine; 6x(13)C labeled L-arginine; (4: 4
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2013-11-23 14:37:53	ID requested
⏵ 1	2015-07-28 09:35:03	announced

Wu X, Renuse S, Sahasrabuddhe NA, Zahari MS, Chaerkady R, Kim MS, Nirujogi RS, Mohseni M, Kumar P, Raju R, Zhong J, Yang J, Neiswinger J, Jeong JS, Newman R, Powers MA, Somani BL, Gabrielson E, Sukumar S, Stearns V, Qian J, Zhu H, Vogelstein B, Park BH, Pandey A, Activation of diverse signalling pathways by oncogenic PIK3CA mutations. Nat Commun, 5():4961(2014) [pubmed]

submitter keyword: PIK3CA, breast cancer, phosphorylation, LS-MS/MS, proteome, mutation

Akhilesh Pandey
contact affiliation	McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, Maryland 21205 USA
contact email	pandey@jhmi.edu
lab head
Akhilesh Pandey
contact affiliation	Institute of Genetic Medicine
contact email	pandey@jhmi.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD000599
     1. Label: PRIDE project
     2. Name: Phosphoproteomic analysis of breast epithelial cells with isogenic knockin of PIK3CA mutations