Immunoaffinity Profiling of Tyrosine Phosphorylation in Cancer Cells

Nat Biotechnol. 2005 Jan;23(1):94-101. doi: 10.1038/nbt1046. Epub 2004 Dec 12.

Abstract

Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • Cell Line
  • Cell Line, Tumor
  • Chromatography, Liquid
  • Gene Expression Regulation, Neoplastic*
  • Humans
  • Jurkat Cells
  • Mass Spectrometry
  • Mice
  • NIH 3T3 Cells
  • Neoplasms / metabolism*
  • Peptides / chemistry
  • Phosphorylation
  • Phosphotyrosine / chemistry
  • Proteomics / methods*
  • Signal Transduction
  • Tyrosine / chemistry*

Substances

  • Peptides
  • Phosphotyrosine
  • Tyrosine