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Quantitative interaction proteomics using mass spectrometry

Nature Methods volume 6, pages 203–205 (2009)Cite this article

Abstract

We present a mass spectrometry–based strategy for the absolute quantification of protein complex components isolated through affinity purification. We quantified bait proteins via isotope-labeled reference peptides corresponding to an affinity tag sequence and prey proteins by label-free correlational quantification using the precursor ion signal intensities of proteotypic peptides generated in reciprocal purifications. We used this method to quantitatively analyze interaction stoichiometries in the human protein phosphatase 2A network.

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Figure 1: Schematic representation of the three main steps of the AP-MS/MS quantification method.
Figure 2: Assessment of the AP-MS/MS quantification method.
Figure 3: A quantitative protein interaction analysis of PP2A.

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Acknowledgements

We thank O. Rinner and M. Beck for helpful discussions, M. Varjosalo for critically reading the manuscript, and the laboratory of E. Ogris (Max F. Perutz Laboratories) for providing the PPME1 antibody. This project has been funded in part by Eidgenössische Technische Hochschule Zurich, and the US National Heart, Lung, and Blood Institute, National Institutes of Health, under contract N01-HV-28179, and the European Union–funded large integrated FP7 project Systems Biology of T-cell Activation. A.W. and R.A. were supported in part by a grant from F. Hoffmann-La Roche Ltd. provided to the Competence Center for Systems Physiology and Metabolic Disease.

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Authors and Affiliations

  1. Institute of Molecular Systems Biology, Swiss Federal Institute of Technology, Eidgenössische Technische Hochschule Zürich, Wolfgang Pauli-Str. 16, Zurich, CH-8093, Switzerland

    Alexander Wepf, Timo Glatter, Alexander Schmidt, Ruedi Aebersold & Matthias Gstaiger

  2. Competence Center for Systems Physiology and Metabolic Diseases, Schafmattstr. 18, Zurich, CH-8093, Switzerland

    Alexander Wepf, Timo Glatter, Alexander Schmidt, Ruedi Aebersold & Matthias Gstaiger

  3. Faculty of Science, University of Zurich, Winterthurerstr. 190, Zurich, CH-8057, Switzerland

    Ruedi Aebersold

  4. Institute of Systems Biology, 1441 North 34th Street, Seattle, 98103, Washington, USA

    Ruedi Aebersold

Authors
  1. Alexander Wepf
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  2. Timo Glatter
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  3. Alexander Schmidt
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  4. Ruedi Aebersold
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  5. Matthias Gstaiger
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Contributions

A.W. performed the experimental and analytical work and wrote the manuscript together with M.G.; T.G. provided the cell lines, and A.S. helped with mass-spectrometry measurements. R.A. and M.G. provided conceptual advice. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Matthias Gstaiger.

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Supplementary Figures 1–3, Supplementary Tables 1–2, Supplementary Results, Supplementary Discussion, Supplementary Methods (PDF 1412 kb)

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Wepf, A., Glatter, T., Schmidt, A. et al. Quantitative interaction proteomics using mass spectrometry. Nat Methods 6, 203–205 (2009). https://doi.org/10.1038/nmeth.1302

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