Open Access

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

  • David R. Stover1, 2, 3Email author,
  • Jennifer Caldwell1, 3,
  • Jarrod Marto1, 3,
  • Karen Root1, 3,
  • Juergan Mestan4,
  • Michael Stumm4,
  • Olga Ornatsky1, 3,
  • Chris Orsi1, 3,
  • Nina Radosevic1, 3,
  • Linda Liao5,
  • Doriano Fabbro4 and
  • Michael F. Moran5
Clinical Proteomics1:11069

DOI: 10.1385/CP:1:1:069

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.

Key Words

Phosphoproteomics proteomics Fourier transform mass spectrometry (FTMS) Epidermal Growth Factor Receptor (EGFR) PKI166

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