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Interlaboratory reproducibility of large-scale human protein-complex analysis by standardized AP-MS

Nature Methods volume 10, pages 307314 (2013) | Download Citation

Abstract

The characterization of all protein complexes of human cells under defined physiological conditions using affinity purification–mass spectrometry (AP-MS) is a highly desirable step in the quest to understand the phenotypic effects of genomic information. However, such a challenging goal has not yet been achieved, as it requires reproducibility of the experimental workflow and high data consistency across different studies and laboratories. We systematically investigated the reproducibility of a standardized AP-MS workflow by performing a rigorous interlaboratory comparative analysis of the interactomes of 32 human kinases. We show that it is possible to achieve high interlaboratory reproducibility of this standardized workflow despite differences in mass spectrometry configurations and subtle sample preparation–related variations and that combination of independent data sets improves the approach sensitivity, resulting in even more-detailed networks. Our analysis demonstrates the feasibility of obtaining a high-quality map of the human protein interactome with a multilaboratory project.

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Acknowledgements

The authors thank A. Nebreda for expert discussion on the biological implications of interactions found, F. Breitwieser and K. Parapatics for support and all laboratory members in Zurich and Vienna for useful discussions. At ETHZ this work was supported by the European Union 7th Framework project PROSPECTS (Proteomics Specification in Space and Time, grant HEALTH-F4-2008-201648), the SystemsX project PhosphonetX and European Research Council advanced grant 'Proteomics v3.0' (grant no. 233226) of the European Union to R.A. and European Union 7th Framework Marie Curie Actions Intra-European Fellowships grant 'Cancer Kinome' (grant no. 236839) to M.V. At CeMM the work was supported by the Austrian Academy of Sciences and Austrian Federal Ministry for Science and Research (Genome Research in Austria projects Austrian Proteomics Platform III (grant no. 820965) to G.S.-F. and Bioinformatics Integration Network III (grant no. 820962) to J.C.).

Author information

Author notes

    • Markku Varjosalo
    • , Roberto Sacco
    •  & Alexey Stukalov

    These authors contributed equally to this work.

Affiliations

  1. Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.

    • Markku Varjosalo
    • , Audrey van Drogen
    • , Simon Hauri
    • , Ruedi Aebersold
    •  & Matthias Gstaiger
  2. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

    • Roberto Sacco
    • , Alexey Stukalov
    • , Melanie Planyavsky
    • , Keiryn L Bennett
    • , Jacques Colinge
    •  & Giulio Superti-Furga
  3. Faculty of Science, University of Zurich, Zurich, Switzerland.

    • Ruedi Aebersold

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Contributions

M.V., R.S., A.v.D., M.P. and S.H. performed experiments, M.G. and G.S.-F. conceived the study and M.V., R.S., K.L.B., J.C., M.G. and G.S.-F. designed experiments. A.S. and J.C. did bioinformatic and statistical data analysis. M.V., R.S., A.S., R.A., K.L.B., J.C., M.G. and G.S.-F. contributed to the discussion of results and participated in manuscript preparation. M.V., R.S., A.S., J.C., M.G. and G.S.-F. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Matthias Gstaiger or Giulio Superti-Furga.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1 and 2, Supplementary Tables 2, 3 and 5 and Supplementary Notes 1 and 2

Zip files

  1. 1.

    Supplementary Data

    Kinase interaction networks: Cytoscape file containing filtered and unfiltered interactomes obtained in the study

Excel files

  1. 1.

    Supplementary Table 1

    Properties of the 32 kinases selected for the study

  2. 2.

    Supplementary Table 4

    Table of full unfiltered AP-MS data

About this article

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DOI

https://doi.org/10.1038/nmeth.2400

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