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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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.).
The authors declare no competing financial interests.
Supplementary Figures 1 and 2, Supplementary Tables 2, 3 and 5 and Supplementary Notes 1 and 2 (PDF 561 kb)
Kinase interaction networks: Cytoscape file containing filtered and unfiltered interactomes obtained in the study (ZIP 1008 kb)
Properties of the 32 kinases selected for the study (XLSX 16 kb)
Table of full unfiltered AP-MS data (XLSX 1688 kb)
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Varjosalo, M., Sacco, R., Stukalov, A. et al. Interlaboratory reproducibility of large-scale human protein-complex analysis by standardized AP-MS. Nat Methods 10, 307–314 (2013). https://doi.org/10.1038/nmeth.2400
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