Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics

Nat Biotechnol. 2004 Sep;22(9):1139-45. doi: 10.1038/nbt1005. Epub 2004 Aug 15.


To study the global dynamics of phosphotyrosine-based signaling events in early growth factor stimulation, we developed a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance. The proteomes of three cell populations were metabolically encoded with different stable isotopic forms of arginine. Each population was stimulated by epidermal growth factor for a different length of time, and tyrosine-phosphorylated proteins and closely associated binders were affinity purified. Arginine-containing peptides occurred in three forms, which were quantified; we then combined two experiments to generate five-point dynamic profiles. We identified 81 signaling proteins, including virtually all known epidermal growth factor receptor substrates, 31 novel effectors and the time course of their activation upon epidermal growth factor stimulation. Global activation profiles provide an informative perspective on cell signaling and will be crucial to modeling signaling networks in a systems biology approach.

Publication types

  • Letter
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Epidermal Growth Factor / pharmacology
  • ErbB Receptors / metabolism*
  • Gene Expression Regulation / drug effects
  • Gene Expression Regulation / physiology*
  • HeLa Cells
  • Humans
  • Isotope Labeling / methods
  • Kinetics
  • Mass Spectrometry / methods*
  • Oxidative Phosphorylation
  • Phosphotyrosine / metabolism*
  • Proteome / metabolism*
  • Proteomics / methods*
  • Signal Transduction / drug effects
  • Signal Transduction / physiology*
  • Time Factors


  • Proteome
  • Phosphotyrosine
  • Epidermal Growth Factor
  • ErbB Receptors