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

Clinical Proteomics volume 1, pages69–80(2004)Cite this article

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.

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Affiliations

  1. MDS Proteomics Inc., 1670 Discovery Dr., Charlottesville, VA

    David R. Stover, Jennifer Caldwell, Jarrod Marto, Karen Root, Olga Ornatsky, Chris Orsi & Nina Radosevic

  2. 25 I Attwell Drive, M9Q 7H4, Toronto, Canada

    David R. Stover, Jennifer Caldwell, Jarrod Marto, Karen Root, Olga Ornatsky, Chris Orsi & Nina Radosevic

  3. Oncology Research, Novartis Pharma AG, CH-4002, Basel, Switzerland

    Juergan Mestan, Michael Stumm & Doriano Fabbro

  4. Oncology Research, Novartis Institutes for Biomedical Research, 02139, Cambridge, MA

    David R. Stover

  5. Banting and Best Department of Medical Research, University of Toronto, 112 College St., M5G IL6, Toronto, ON, Canada

    Linda Liao & Michael F. Moran

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  1. David R. Stover
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  2. Jennifer Caldwell
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  3. Jarrod Marto
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  4. Karen Root
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  5. Juergan Mestan
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  6. Michael Stumm
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  7. Olga Ornatsky
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  8. Chris Orsi
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  9. Nina Radosevic
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  10. Linda Liao
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  11. Doriano Fabbro
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  12. Michael F. Moran
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Correspondence to David R. Stover.

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Stover, D.R., Caldwell, J., Marto, J. et al. Differential phosphoprofiles of EGF and EGFR kinase inhibitor-treated human tumor cells and mouse xenografts. Clin Proteom 1, 69–80 (2004). https://doi.org/10.1385/CP:1:1:069

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Key Words

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

Clinical Proteomics

ISSN: 1559-0275