Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics


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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Figure 1: Determining the activation profile of the EGFR phosphotyrosine-dependent proteome.
Figure 2: Western blot analysis of selected EGFR effectors.
Figure 3: Activation profiles of different categories of EGFR signaling effectors, derived from mass spectrometric data as shown in Figure 1b and documented in Table 1.


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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).

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Correspondence to Matthias Mann.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Variability of kinetic data. (PDF 44 kb)

Supplementary Table 1

Proteins activated upon EGF stimulation. (PDF 111 kb)

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