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The CRAPome: a contaminant repository for affinity purification–mass spectrometry data

Abstract

Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. Fortunately, negative controls are largely bait independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the contaminant repository for affinity purification (the CRAPome) and describe its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.

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Figure 1: The CRAPome at a glance.
Figure 2: Composition of the CRAPome (human data).
Figure 3: Scoring functions in the CRAPome illustrated on a four-bait data set (MEPCE, EIF4A2, WASL and RAF1; eight experiments).
Figure 4: Use of the FC-B score to recover true interacting partners for ORC2L.

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Acknowledgements

We wish to thank G.I. Chen, M. Mullin, M.J. Kean, T.M. Greco, T. Srikumar and Y.-C. Tsai for contributing published data and S. Saha and J.-E. Dazard for constructive comments. We thank A. Stefanovic, M. Planyavsky, A. Stukalov, A.J. Guise, A.C. Müller, A. Pichlmair, B. Larsen, C. Knoll, C.L. Baumann, E.L. Rudashevskaya, F. Grebien, F.P. Breitwieser, H.G. Budayeva, J.W. Bigenzahn, M. Bruckner, M. Licciardello, M.L. Huber, M. Tucholska, N. Venturini, O. Rocks, O. Stein, P. Joshi, R. Giambruno, R. Sacco, S. Zhang, T. Stasyk and V. Nguyen for help with sample analysis.

This work was supported by grants from the US National Institutes of Health (NIH 5R01GM94231 to A.-C.G. and A.I.N.; DP1DA026192 and HL112618-01 to I.M.C.), the Canadian Institutes of Health Research (CIHR MOP-84314 to A.-C.G.; MOP-82851 to B.C.), the government of Ontario via a Global Leadership Round in Genomics and Life Sciences (T.P. and A.-C.G.), the Austrian Academy of Sciences (K.L.B., J.C. and G.S.-F.), the Austrian Federal Ministry for Science and Research (Gen-Au projects, APP-III and BIN-III; K.L.B. and G.S.-F., no. 820965; J.C. and K.L.B., no. 820962), the European Research Council (G.S.-F.; ERC-2009-AdG-250179-i-FIVE), the Austrian Science Fund FWF (G.S.-F., J.C. and K.L.B.; P24321-B21 and P22282-B11), the European Molecular Biology Organization long-term fellowship (G.S.-F., J.C. and K.L.B.; ATLF463-2008), The Netherlands Proteomics Center (T.Y.L., V.A.H., S.M. and A.J.R.H.), the European Union 7th Framework Program (PRIME-XS project, grant no. 262067; T.Y.L., V.A.H., S.M., R.A. and A.J.R.H.), the Stowers Institute for Medical Research, and the Human Frontier Science Program (RGY0079/2009-C to I.M.C.). A.-C.G. is the Canada Research Chair in Functional Proteomics and the Lea Reichmann Chair in Cancer Proteomics; B.R. is the Canada Research Chair in Proteomics and Molecular Medicine. R.M.E. acknowledges salary support from the Cleveland Foundation and NIH 1R21 CA16006001A1. J.-P.L. and R.D.B. were supported by CIHR postdoctoral awards. N.A.S.-D. was supported by a TD Bank postdoctoral fellowship.

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Contributions

D.M., Z.W., A.I.N. and A.-C.G. designed the CRAPome structure and interface; D.M. and Z.W. implemented the system; D.M. and A.I.N. created the scoring scheme with help from H.C. and A.-C.G.; R.M.E., A.-C.G. and A.I.N. initiated the project; D. Fermin helped with processing the data; B.R., A.L.C., N.A.S.-D. and J.-P.L. tested the interface and contributed to editing the user manuals; A.L.C., N.A.S.-D., J.-P.L., W.H.D., T.L., Y.V.M., S.H., M.E.S., T.Y.L., V.A.H., R.D.B., N.C.H., A.a.-H., A.B., D. Faubert, R.M.E., I.M.C., K.L.B. and A.-C.G. provided mass spectrometry data to the CRAPome and/or annotated data in the repository; Z.-Y.L., B.G.B. and M. Goudreault contributed the test benchmark data set; T.P., D.D., B.C., R.A., G.S.-F., J.C., A.J.R.H., M. Gstaiger, S.M., I.M.C., K.L.B., M.P.W. and A.-C.G. supervised trainees and were responsible for data generation across the different research groups; H.C., B.R., I.M.C., K.L.B., M.P.W. and R.M.E. provided critical comments throughout the project; A.-C.G. and A.I.N. codirected the project and analyzed and annotated data; A.I.N., A.-C.G. and D.M. wrote the manuscript and the user manuals with help from B.R.

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Correspondence to Anne-Claude Gingras or Alexey I Nesvizhskii.

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Mellacheruvu, D., Wright, Z., Couzens, A. et al. The CRAPome: a contaminant repository for affinity purification–mass spectrometry data. Nat Methods 10, 730–736 (2013). https://doi.org/10.1038/nmeth.2557

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