PXD003544

PXD003544 is an original dataset announced via ProteomeXchange.

Title	Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and their interacting stromal fibroblast cells
Description	Communication and interaction between carcinoma associated fibroblast (CAF) cells and tumor epithelium cells plays a critical role in cancer initiation and progression. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. In this study, we employed a SILAC labeling based co-culture system coupled with mass spectrometry analysis to identify and quantify the early phospho-signaling transductions induced by the crosstalk between CAFs and tumor cells. We performed three-state SILAC labeling for MDA-MB-231 and 82T. 9 million light labeled 82-L cells were co-cultured with medium labeled MDA-MB-231-M cells for 30 minutes. 9 million heavy labeled 82T-H cells were individually cultured in heavy SILAC media with for 30 minutes. Both individually cultured and co-cultured cells were quickly harvested with 9 M urea lysis buffer and mixed together. Mixed lysates were sonicated, reduced, alkylated and digested by trypsin. Tryptic peptides were desalted using SepPak C18 cartridge and followed by phosphotyrosine peptide enrichment using immunoaffinity purification with pY100 antibody. The enriched phosphopeptides were analyzed using a reverse-phase liquid chromatography system interfaced with an LTQ-Orbitrap Velos mass spectrometer. The Proteome Discoverer (v2.0; Thermo Fisher Scientific) suite was used for quantitation and database searches. The tandem mass spectrometry data were searched using SEQUEST search algorithm against a Human RefSeq database supplemented with frequently observed contaminants. The probability of phosphorylation sites was determined from the PhosphoRS algorithm.
HostingRepository	PRIDE
AnnounceDate	2018-10-26
AnnouncementXML	Submission_2018-10-26_05:18:36.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	phosphorylated residue
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2016-01-29 00:30:47	ID requested
⏵ 1	2018-10-26 05:18:38	announced

Wu X, Zahari MS, Renuse S, Sahasrabuddhe NA, Chaerkady R, Kim MS, Fackler MJ, Stampfer M, Gabrielson E, Sukumar S, Pandey A, Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts. Clin Proteomics, 15():21(2018) [pubmed]

curator keyword: Biomedical

submitter keyword: LC/MS-MS, breast cancer, fibroblast, tumor, epithelial cell, kinase, phosphorylation

Akhilesh Pandey
contact affiliation	Professor of Biological Chemistry, Oncology and Pathology, Johns Hopkins University
contact email	pandey@jhmi.edu
lab head
Akhilesh Pandey
contact affiliation	Johns Hopkins University
contact email	pandey@jhmi.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD003544
     1. Label: PRIDE project
     2. Name: Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and their interacting stromal fibroblast cells