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Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition

Abstract

Characterizing changes in protein-protein interactions associated with sequence variants (e.g., disease-associated mutations or splice forms) or following exposure to drugs, growth factors or hormones is critical to understanding how protein complexes are built, localized and regulated. Affinity purification (AP) coupled with mass spectrometry permits the analysis of protein interactions under near-physiological conditions, yet monitoring interaction changes requires the development of a robust and sensitive quantitative approach, especially for large-scale studies in which cost and time are major considerations. We have coupled AP to data-independent mass spectrometric acquisition (sequential window acquisition of all theoretical spectra, SWATH) and implemented an automated data extraction and statistical analysis pipeline to score modulated interactions. We used AP-SWATH to characterize changes in protein-protein interactions imparted by the HSP90 inhibitor NVP-AUY922 or melanoma-associated mutations in the human kinase CDK4. We show that AP-SWATH is a robust label-free approach to characterize such changes and propose a scalable pipeline for systems biology studies.

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Figure 1: AP-SWATH pipeline.
Figure 2: AP-SWATH for scoring protein interactions.
Figure 3: Selected biological samples.
Figure 4: Identification of differential interactomes for CDK4 cancer-associated mutants.
Figure 5: Use of AP-SWATH to probe drug-modulated interactions.

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Acknowledgements

We thank L. Taylor for input and initial SWATH data analysis, E. Polvi for help with subcloning, K. Colwill (Lunenfeld-Tanenbaum Research Institute) for the construction of pDEST-5′Triple-Flag-pcDNA5-FRT-TO and B. Raught for critical reading of the manuscript. The website for the supplementary material was designed by G. Liu and J.P. Zhang. This work was supported by a Venture Sinai award to A.-C.G.; the Canadian Institute of Health Research (to A.-C.G.; MOP-84314); the US National Institutes of Health (to A.-C.G.; 5R01GM94231); the Ontario Research Fund via a Global Leadership Award Round 2 (to T.P. and A.-C.G.).; the European Research Council (to R.A.; #ERC-2008-AdG 233226); SystemsX.ch, the Swiss Initiative for Systems Biology (to R.A.); the US National Human Genome Research Institute (to M.V.; R01HG001715 and P50HG004233) and the US National Cancer Institute (to M.V.; R33CA132073); a Canada Research Chair in Functional Proteomics and the Lea Reichmann Chair in Cancer Proteomics (to A.-C.G.).; and postdoctoral awards from the Canadian Institutes of Health Research and the Canadian National Sciences and Engineering Research Council postdoctoral award (to J.-P.L.).

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Authors

Contributions

J.-P.L. generated all CDK4 samples and performed validation experiments; G.I. developed the pipeline for the normalization and fold-change calculation and performed statistical analysis; A.L.C. generated all GRK6 samples and performed validation experiments; M.T. performed LUMIER analysis and provided constructs; Z.-Y.L. prepared samples for mass spectrometry; B.L. and S.T. performed mass spectrometric measurements and iTRAQ data analysis; Q.Z. and M.V. provided initial constructs and input on the project; S.L. supervised M.T., and T.P. cosupervised J.-P.L.; R.A., R.B. and S.T. co-developed the SWATH approach; J.-P.L., A.L.C., B.L., A.-C.G., G.I. and S.T. analyzed the SWATH data; A.-C.G. wrote the manuscript with input from all authors; A.-C.G. conceived of the study and directed the project.

Corresponding authors

Correspondence to Stephen Tate or Anne-Claude Gingras.

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Competing interests

G.I., R.B. and S.T. are employees of AB Sciex. AB Sciex provided support for the Ontario Research Fund grant to T.P. and A.-C.G.

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Lambert, JP., Ivosev, G., Couzens, A. et al. Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition. Nat Methods 10, 1239–1245 (2013). https://doi.org/10.1038/nmeth.2702

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