Differential phosphoprofiles of EGF and EGFR kinase inhibitor-treated human tumor cells and mouse xenografts

Clinical Proteomics

Michael F. Moran⁴  Olga Ornatsky²  Karen Root²  Linda Liao⁴  Jarrod Marto²  Jennifer Caldwell²  Juergan Mestan³  David R. Stover¹  Nina Radosevic²  Doriano Fabbro³  Chris Orsi²  Michael Stumm³ 

[1] MDS Proteomics Inc., Charlottesville, Oncology Research, Novartis Institutes for Biomedical Research, Cambridge, Toronto, CanadaMDS Proteomics Inc., Charlottesville, MDS Proteomics Inc., Charlottesville, Oncology Research, Novartis Institutes for Biomedical Research, Cambridge, Toronto, CanadaOncology Research, Novartis Institutes for Biomedical Research, Cambridge, MDS Proteomics Inc., Charlottesville, Oncology Research, Novartis Institutes for Biomedical Research, Cambridge, Toronto, CanadaToronto, CanadaMDS Proteomics Inc., Charlottesville, Oncology Research, Novartis Institutes for Biomedical Research, Cambridge, Toronto, Canada;MDS Proteomics Inc., Charlottesville, Toronto, CanadaMDS Proteomics Inc., Charlottesville, MDS Proteomics Inc., Charlottesville, Toronto, CanadaToronto, CanadaMDS Proteomics Inc., Charlottesville, Toronto, Canada;Oncology Research, Novartis Pharma AG, Basel, SwitzerlandOncology Research, Novartis Pharma AG, Basel, SwitzerlandOncology Research, Novartis Pharma AG, Basel, Switzerland;Banting and Best Department of Medical Research, University of Toronto, Toronto, CanadaBanting and Best Department of Medical Research, University of Toronto, Toronto, CanadaBanting and Best Department of Medical Research, University of Toronto, Toronto, Canada

关键词: Phosphoproteomics;    proteomics;    Fourier transform mass spectrometry (FTMS);    Epidermal Growth Factor Receptor (EGFR);    PKI166;   

DOI  :  10.1385/CP:1:1:069

来源: Humana Press Inc

【 摘 要 】

Abstract

The purpose of this phospho-proteomics study was to demonstrate the broad analysis of cellular protein phosphorylation in cells and tissue as a means to monitor changes in cellular states. As a cancer model, human tumor-derived A431 cells known to express the epidermal growth factor receptor (EGFR) were grown as cell cultures or xenograft tumors in mice. The cells and tumor-bearing animals were subjected to treatments including the EGFR-directed protein kinase inhibitor PK166 and/or EGF stimulation. Whole cell/tissue protein extracts were converted to peptides by using trypsin, and phosphorylated peptides were purified by an affinity capture method. Peptides and phosphorylation sites were characterized and quantified by using a combination of tandem mass spectroscopy (MS) and Fourier transform MS instrumentation (FTMS). By analyzing roughly 10⁶ cell equivalents, 780 unique phosphopeptides from approx 450 different proteins were characterized. Only a small number of these phosphorylation sites have been described previously in literature. Although a targeted analysis of the EGFR pathway was not a specific aim of this study, 22 proteins known to be associated with EGFR signaling were identified. Fifty phosphopeptides were found changed in abundance as a function of growth factor or drug treatment including novel sites of phosphorylation on the EGFR itself. These findings demonstrate the feasibility of using phospho-proteomics to determine drug and disease mechanisms, and as a measure of drug target modulation in tissue.

【 授权许可】

Unknown