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Proteomics strategy for quantitative protein interaction profiling in cell extracts

Nature Methods volume 6, pages 741–744 (2009)Cite this article

Abstract

We report a proteomics strategy to both identify and quantify cellular target protein interactions with externally introduced ligands. We determined dissociation constants for target proteins interacting with the ligand of interest by combining quantitative mass spectrometry with a defined set of affinity purification experiments. We demonstrate the general utility of this methodology in interaction studies involving small-molecule kinase inhibitors, a tyrosine-phosphorylated peptide and an antibody as affinity ligands.

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Figure 1: Assessment of target-specific dissociation constants by quantitative chemical proteomics.
Figure 2: Cellular kinase panel for quantitative inhibitor profiling.
Figure 3: Quantitative peptide profiling and immunoprecipitation analysis.

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Acknowledgements

We thank A. Ullrich for his support of the present study with his doctorate student M.B. and funding from the Department of Molecular Biology, Max Planck Institute of Biochemistry, Martinsried and M. Mann for support. K.S. was supported by research fellowships from the Max Planck Society and the Alexander von Humboldt Foundation. This work was supported by a grant from the Deutsche Krebshilfe.

Author information

Author notes

  1. Kirti Sharma & Jesper V Olsen

    Present address: Present addresses: ITC Research and Development Centre, Survey Nos 230 and 243, Turkapally Village, Hyderabad, India (K.S.) and Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark (J.V.O.).,

  2. Kirti Sharma, Christoph Weber and Michaela Bairlein: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology, Cell Signaling Group, Max Planck Institute of Biochemistry, Martinsried, Germany

    Kirti Sharma, Christoph Weber & Henrik Daub

  2. Department of Molecular Biology, Max Planck Institute of Biochemistry, Martinsried, Germany

    Michaela Bairlein

  3. Vichem Chemie Ltd., Budapest, Hungary

    Zoltán Greff & György Kéri

  4. Pathobiochemistry Research Group of the Hungarian Academy of Science, Semmelweis University, Budapest, Hungary

    György Kéri

  5. Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany

    Jürgen Cox & Jesper V Olsen

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Contributions

K.S., C.W. and M.B. contributed to the experimental design, performed experiments and analyzed data. Z.G. and G.K. prepared small-molecule inhibitors. J.C. provided tools for data analysis and contributed to data analysis. J.V.O. provided mass spectrometry expertise and contributed to the experimental design and data analysis. H.D. designed the study, performed data analysis and wrote the paper.

Corresponding author

Correspondence to Henrik Daub.

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Competing interests

A patent application of the proteomics approach described in this study has been filed and has been licensed to Kinaxo Biotechnologies GmbH, Martinsried, Germany. H.D. is a shareholder of Kinaxo Biotechnologies GmbH, and H.D., J.V.O. and J.C. have consultancy agreements with Kinaxo Biotechnologies GmbH. Z.G. and G.K. are shareholders and employees of Vichem Chemie Ltd. Budapest, Hungary.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1–7, Supplementary Note and Supplementary Discussion (PDF 594 kb)

Supplementary Data 1

Protein groups data of AX14596 and gefitinib experiments (XLS 2676 kb)

Supplementary Data 2

Peptide evidences data of AX14596 and gefitinib experiments (XLS 25918 kb)

Supplementary Data 3

Quantification results and Kd and IC50 values for specific cellular targets of AX14596 and gefitinib (XLS 55 kb)

Supplementary Data 4

Protein groups data of VI16742 and SB203580 experiments (XLS 2346 kb)

Supplementary Data 5

Peptide evidences data of VI16742 and SB203580 experiments (XLS 22794 kb)

Supplementary Data 6

Quantification results and Kd and IC50 values for specific cellular targets of VI16742 and SB203580 (XLS 234 kb)

Supplementary Data 7

Protein groups data of IRS4-pY921 peptide interaction experiments (XLS 3379 kb)

Supplementary Data 8

Peptide evidences data of IRS4-pY921 peptide interaction experiments (XLS 16562 kb)

Supplementary Data 9

Quantification results and Kd values for specific cellular interactors of IRS4-pY921 peptide (XLS 282 kb)

Supplementary Data 10

Protein groups data of EGFR immunoprecipitation experiments (XLS 1214 kb)

Supplementary Data 11

Peptide evidences data of EGFR immunoprecipitation experiments (XLS 4082 kb)

Supplementary Data 12

Quantification results for specific cellular interactors of the EGFR (XLS 22 kb)

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Sharma, K., Weber, C., Bairlein, M. et al. Proteomics strategy for quantitative protein interaction profiling in cell extracts. Nat Methods 6, 741–744 (2009). https://doi.org/10.1038/nmeth.1373

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