Dataset Information

Phosphoproteomic analysis of breast epithelial cells with isogenic knockin of PIK3CA mutations

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD000599 | Pride | 2015-07-28

REPOSITORIES: Pride

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			Action	DRS
	120110_PandeyA_WX_Ex9A_D1.raw	Raw
	120110_PandeyA_WX_Ex9A_D10.raw	Raw
	120110_PandeyA_WX_Ex9A_D11.raw	Raw
	120110_PandeyA_WX_Ex9A_D12.raw	Raw
	120110_PandeyA_WX_Ex9A_D12_r.raw	Raw

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Publications

Activation of diverse signalling pathways by oncogenic PIK3CA mutations.

Wu Xinyan X Renuse Santosh S Sahasrabuddhe Nandini A NA Zahari Muhammad Saddiq MS Chaerkady Raghothama R Kim Min-Sik MS Nirujogi Raja S RS Mohseni Morassa M Kumar Praveen P Raju Rajesh R Zhong Jun J Yang Jian J Neiswinger Johnathan J Jeong Jun-Seop JS Newman Robert R Powers Maureen A MA Somani Babu Lal BL Gabrielson Edward E Sukumar Saraswati S Stearns Vered V Qian Jiang J Zhu Heng H Vogelstein Bert B Park Ben Ho BH Pandey Akhilesh A

Nature communications 20140923

The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing 'driver' oncogenic mutations of PIK3CA to dissect the signalling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream ...[more]

PMID: 25247763

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