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.
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We thank other members of our laboratory for help and fruitful discussions. Peter Mortensen is acknowledged for help with the bioinformatic analysis and Carmen de Hoog and Leonard Foster for critical reading of the manuscript. Work in the Center for Experimental BioInformatics (CEBI) is supported by a generous grant by the Danish National Research foundation and by the 6th Framework Program of the European Union (Interaction Proteome).
The authors declare no competing financial interests.
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