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Quantitative proteomic assessment of very early cellular signaling events

Nature Biotechnology volume 25, pages 566–568 (2007)Cite this article

Abstract

Technical limitations have prevented proteomic analyses of events occurring less than 30 s after signal initiation. We developed an automated, continuous quench-flow system allowing quantitative proteomic assessment of very early cellular signaling events (qPACE) with a time resolution of 1 s. Using this technique, we determined that autophosphorylation of the epidermal growth factor receptor occurs within 1 s after ligand stimulation and is followed rapidly by phosphorylation of the downstream signaling intermediates Src homologous and collagen-like protein and phospholipase C gamma 1.

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Figure 1: qPACE-set up.
Figure 2: Activation profiles of phosphopeptides.

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Acknowledgements

We thank all members of the Center for Experimental BioInformatics (CEBI) for help and fruitful discussions, especially Mogens Nielsen, Morten Kirkegaard and Peter Mortensen. TiO2 spheres were a kind gift from GL Sciences. J.D. is supported by the European Molecular Biology Organization (long-term fellowship). CEBI is supported by a generous grant from the Danish National Research Foundation.

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Author notes

  1. Joern Dengjel and Vyacheslav Akimov: These authors contributed equally to the manuscript.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Center of Experimental BioInformatics, University of Southern Denmark, Odense, Campusvej 55, Odense, DK-5230, Denmark

    Joern Dengjel, Vyacheslav Akimov, Jakob Bunkenborg, Blagoy Blagoev & Jens S Andersen

  2. Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Am Klopferspitz 18, Martinsried, D-82152, Germany

    Jesper V Olsen & Matthias Mann

Authors
  1. Joern Dengjel
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  2. Vyacheslav Akimov
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  3. Jesper V Olsen
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  4. Jakob Bunkenborg
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  5. Matthias Mann
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  6. Blagoy Blagoev
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  7. Jens S Andersen
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Contributions

J.D. and V.A.: Figures 1 and 2, Supplementary Figures 1, 2 and 3, cell culture, stimulation experiments, western blot analyses and MS analyses. J.O. and J.B.: Figure 2a and MS analyses. M.M., B.B. and J.S.A.: experiment design, result analysis and writing of the manuscript.

Corresponding authors

Correspondence to Blagoy Blagoev or Jens S Andersen.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Phosphosite-specific EGFR western blots of cells stimulated at 37° C. (PDF 70 kb)

Supplementary Fig. 2

Intensity ratios of unphosphorylated counterparts of detected EGFR phosphopeptides. (PDF 41 kb)

Supplementary Fig. 3

Microcarrier HeLa cell culture stimulated with EGF using the qPACE set-up. (PDF 119 kb)

Supplementary Methods (PDF 81 kb)

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Dengjel, J., Akimov, V., Olsen, J. et al. Quantitative proteomic assessment of very early cellular signaling events. Nat Biotechnol 25, 566–568 (2007). https://doi.org/10.1038/nbt1301

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