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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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.

Author information

Author notes

    • Rob M Ewing

    Present address: Centre for Biological Sciences, University of Southampton, Southampton, UK.

Affiliations

  1. Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA.

    • Dattatreya Mellacheruvu
    • , Damian Fermin
    •  & Alexey I Nesvizhskii
  2. Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA.

    • Dattatreya Mellacheruvu
    • , Zachary Wright
    •  & Alexey I Nesvizhskii
  3. Centre for Systems Biology, Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario, Canada.

    • Amber L Couzens
    • , Jean-Philippe Lambert
    • , Nicole A St-Denis
    • , Richard D Bagshaw
    • , Abdallah al-Hakim
    • , Wade H Dunham
    • , Marilyn Goudreault
    • , Zhen-Yuan Lin
    • , Beatriz Gonzalez Badillo
    • , Tony Pawson
    • , Daniel Durocher
    •  & Anne-Claude Gingras
  4. Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA.

    • Tuo Li
    • , Yana V Miteva
    •  & Ileana M Cristea
  5. Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.

    • Simon Hauri
    • , Ruedi Aebersold
    •  & Matthias Gstaiger
  6. Stowers Institute for Medical Research, Kansas City, Missouri, USA.

    • Mihaela E Sardiu
    •  & Mike P Washburn
  7. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.

    • Teck Yew Low
    • , Vincentius A Halim
    • , Albert J R Heck
    •  & Shabaz Mohammed
  8. Netherlands Proteomics Center, Utrecht, The Netherlands.

    • Teck Yew Low
    • , Vincentius A Halim
    • , Albert J R Heck
    •  & Shabaz Mohammed
  9. Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands.

    • Vincentius A Halim
  10. Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Radboud University, Nijmegen, The Netherlands.

    • Nina C Hubner
  11. Institut de Recherches Cliniques de Montréal, Montreal, Quebec, Canada.

    • Annie Bouchard
    • , Denis Faubert
    •  & Benoit Coulombe
  12. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

    • Wade H Dunham
    • , Tony Pawson
    • , Daniel Durocher
    •  & Anne-Claude Gingras
  13. Département de Biochimie, Université de Montréal, Montreal, Quebec, Canada.

    • Benoit Coulombe
  14. Faculty of Science, University of Zurich, Zurich, Switzerland.

    • Ruedi Aebersold
  15. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

    • Giulio Superti-Furga
    • , Jacques Colinge
    •  & Keiryn L Bennett
  16. Saw Swee Hock School of Public Health, National University of Singapore, Singapore.

    • Hyungwon Choi
  17. Department of Chemistry, University of Oxford, Oxford, UK.

    • Shabaz Mohammed
  18. Department of Biochemistry, University of Oxford, Oxford, UK.

    • Shabaz Mohammed
  19. Department of Pathology & Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, USA.

    • Mike P Washburn
  20. Ontario Cancer Institute, Toronto, Ontario, Canada.

    • Brian Raught
  21. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

    • Brian Raught
  22. Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio, USA.

    • Rob M Ewing

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Anne-Claude Gingras or Alexey I Nesvizhskii.

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DOI

https://doi.org/10.1038/nmeth.2557

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