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Quantitative multiplexed profiling of cellular signaling networks using phosphotyrosine-specific DNA-tagged SH2 domains

Nature Methods volume 3, pages 737–744 (2006)Cite this article

Abstract

Deciphering global signaling networks is of great importance for the detailed understanding of cellular signaling processes controlling many important biological functions. Among signaling processes, tyrosine phosphorylation has a central role. At present, adequate techniques for the global characterization of the tyrosine phosphoproteome are lacking, particularly for the analysis of small amounts of protein. By combining the power of PCR amplification with the unique properties of Src homology region 2 (SH2) domains to specifically recognize tyrosine-phosphorylated proteins, we developed a new proteomic approach, termed oligonucleotide-tagged multiplex assay (OTM). For OTM, multiple SH2 domains are labeled by domain-specific oligonucleotide tags, applied as probes to complex protein mixtures in a multiplex reaction and phosphotyrosine-specific interactions are quantified by PCR. Using OTM we reproducibly quantified differential states of tyrosine phosphorylation with high sensitivity and specificity in small amounts of whole cellular extracts as demonstrated for various tumor cell lines and human leukemia samples.

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Figure 1: Principle of the OTM assay.
Figure 2: Evaluation of assay sensitivity, specificity and reproducibility.
Figure 3: Quantitative SH2 profiling of the human PDGFβ receptor by OTM.
Figure 4: Quantitative OTM profiles of different tumor cell lines at different states of activation.
Figure 5: Quantitative SH2 profiling of different types of human leukemia by OTM.

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References

  1. Hunter, T. Signaling–2000 and beyond. Cell 100, 113–127 (2000).

    Article  CAS  Google Scholar 

  2. Ren, R. Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia. Nat. Rev. Cancer 5, 172–183 (2005).

    Article  CAS  Google Scholar 

  3. Hynes, N.E. & Lane, H.A. ERBB receptors and cancer: the complexity of targeted inhibitors. Nat. Rev. Cancer 5, 341–354 (2005).

    Article  CAS  Google Scholar 

  4. Arteaga, C.L. & Baselga, J. Tyrosine kinase inhibitors: why does the current process of clinical development not apply to them? Cancer Cell 5, 525–531 (2004).

    Article  CAS  Google Scholar 

  5. Gilliland, D.G. & Griffin, J.D. The roles of FLT3 in hematopoiesis and leukemia. Blood 100, 1532–1542 (2002).

    Article  CAS  Google Scholar 

  6. Machida, K., Mayer, B.J. & Nollau, P. Profiling the global tyrosine phosphorylation state. Mol. Cell. Proteomics 2, 215–233 (2003).

    Article  CAS  Google Scholar 

  7. Johnson, S.A. & Hunter, T. Kinomics: methods for deciphering the kinome. Nat. Methods 2, 17–25 (2005).

    Article  CAS  Google Scholar 

  8. Salomon, A.R. et al. Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry. Proc. Natl. Acad. Sci. USA 100, 443–448 (2003).

    Article  CAS  Google Scholar 

  9. Rush, J. et al. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol. 23, 94–101 (2005).

    Article  CAS  Google Scholar 

  10. Blagoev, B. et al. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat. Biotechnol. 21, 315–318 (2003).

    Article  CAS  Google Scholar 

  11. Tao, W.A. et al. Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry. Nat. Methods 2, 591–598 (2005).

    Article  CAS  Google Scholar 

  12. Irish, J.M. et al. Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell 118, 217–228 (2004).

    Article  CAS  Google Scholar 

  13. Nollau, P. & Mayer, B.J. Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding. Proc. Natl. Acad. Sci. USA 98, 13531–13536 (2001).

    Article  CAS  Google Scholar 

  14. Pawson, T. Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 116, 191–203 (2004).

    Article  CAS  Google Scholar 

  15. Liu, B.A. et al. The Human and Mouse Complement of SH2 Domain Proteins-Establishing the Boundaries of Phosphotyrosine Signaling. Mol. Cell 22, 851–868 (2006).

    Article  Google Scholar 

  16. Jones, R.B., Gordus, A., Krall, J.A. & Macbeath, G. A quantitative protein interaction network for the ErbB receptors using protein microarrays. Nature 439, 168–174 (2006).

    Article  CAS  Google Scholar 

  17. Stagljar, I. Finding partners: emerging protein interaction technologies applied to signaling networks. Sci. STKE 2003, pe56 (2003).

    PubMed  Google Scholar 

  18. Papin, J.A., Hunter, T., Palsson, B.O. & Subramaniam, S. Reconstruction of cellular signalling networks and analysis of their properties. Nat. Rev. Mol. Cell Biol. 6, 99–111 (2005).

    Article  CAS  Google Scholar 

  19. Yaffe, M.B. Phosphotyrosine-binding domains in signal transduction. Nat. Rev. Mol. Cell Biol. 3, 177–186 (2002).

    Article  CAS  Google Scholar 

  20. Sano, T., Smith, C.L. & Cantor, C.R. Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Science 258, 120–122 (1992).

    Article  CAS  Google Scholar 

  21. Niemeyer, C.M., Adler, M. & Wacker, R. Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification. Trends Biotechnol. 23, 208–216 (2005).

    Article  CAS  Google Scholar 

  22. Hunter, T. Protein modification: phosphorylation on tyrosine residues. Curr. Opin. Cell Biol. 1, 1168–1181 (1989).

    Article  CAS  Google Scholar 

  23. Mayer, B.J., Jackson, P.K. & Baltimore, D. The noncatalytic src homology region 2 segment of abl tyrosine kinase binds to tyrosine-phosphorylated cellular proteins with high affinity. Proc. Natl. Acad. Sci. USA 88, 627–631 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Erich und Gertrud Roggenbuck-Stiftung, Hamburg, Germany (to P.N). Assay development was supported by the US National Institutes of Health grant CA107785 given to B.J.M. and P.N.; J.T. was supported by the Deutsche Forschungsgemeinschaft (SFB470). The authors gratefully acknowledge the support of C. Wagener (University Medical Center Hamburg-Eppendorf). We are very thankful to A. Hansen and B. Klampe for technical assistance, U. zur Stadt and E. Groh (University Medical Center Hamburg-Eppendorf) for access and assistance with the Light Cycler Instrument and H. Pospisil (University Hamburg) for help with the statistical evaluation. We are particularly grateful to H. Sander for assistance during analysis of patient samples.

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Authors and Affiliations

  1. Department of Clinical Chemistry, Center of Clinical Pathology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246, Hamburg, Germany

    Kevin Dierck, Anja Voigt & Peter Nollau

  2. Department of Genetics and Developmental Biology, Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, 06030, Connecticut, USA

    Kazuya Machida & Bruce J Mayer

  3. Institute of Organic Chemistry, University Hamburg, Martin Luther King Platz 6, Hamburg, 20146, Germany

    Julian Thimm

  4. Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Martinstr. 52, Hamburg, 20246, Germany

    Martin Horstmann

  5. Department of Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Martinstr. 52, Hamburg, 20246, Germany

    Walter Fiedler

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  1. Kevin Dierck
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  7. Bruce J Mayer
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Contributions

K.M., generation of SH2 probes; A.V., functional testing of SH2 probes; J.T., atomic force microscopy; M.H., data analyses and statistical evaluation; W.F., investigations on clinical samples; B.J.M., assay development and evaluation.

Corresponding author

Correspondence to Peter Nollau.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Comparable efficiency of PCR amplification of the five oligonucleotide tags. (PDF 132 kb)

Supplementary Fig. 2

Validation of PCR-ELISA. (PDF 75 kb)

Supplementary Fig. 3

Characterization of OTM probes. (PDF 682 kb)

Supplementary Fig. 4

Confirmation of results of PCR-ELISA by quantitative real-time PCR. (PDF 137 kb)

Supplementary Table 1

List of oligonucleotides. (PDF 34 kb)

Supplementary Table 2

Patient characteristics. (PDF 34 kb)

Supplementary Data

Statistical evaluation of OTM data. (PDF 70 kb)

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Dierck, K., Machida, K., Voigt, A. et al. Quantitative multiplexed profiling of cellular signaling networks using phosphotyrosine-specific DNA-tagged SH2 domains. Nat Methods 3, 737–744 (2006). https://doi.org/10.1038/nmeth917

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