Abstract

We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems. However, there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners. Here, we present a screening method that comprehensively explores the parameters affecting the stability of interactions in affinity-captured complexes, enabling the discovery of physiological binding partners in unparalleled detail. We have implemented this screen on several macromolecular complexes from a variety of organisms, revealing novel profiles for even well-studied proteins. Our approach is robust, economical and automatable, providing inroads to the rigorous, systematic dissection of cellular interactomes.

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Acknowledgements

We thank The Rockefeller University High Energy Physics Instrument Shop for diligence in custom apparatus design and fabrication; X. Wang for assistance with MS data analysis; and members of the Chait, Jensen and Rout laboratories for help and discussion. I. Poser and A. Hyman (Max Planck Institute of Molecular Biology and Genetics, Dresden) provided the RBM7-LAP cell line. This work was funded by the US National Institutes of Health (NIH) grant nos. U54 GM103511 and P41 GM109824 (J.D.A., B.T.C. and M.P.R.), P50 GM076547 (J.D.A.) and P41 GM103314 (B.T.C.); the Lundbeck Foundation (to T.H.J. and J.L.) and the Danish National Research Foundations (to T.H.J.).

Author information

Author notes

    • Loren E Hough

    Present address: Department of Physics and BioFrontiers Institute, University of Colorado, Boulder, Colorado, USA.

Affiliations

  1. Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York, USA.

    • Zhanna Hakhverdyan
    • , Michal Domanski
    • , Loren E Hough
    • , Michael P Rout
    •  & John LaCava
  2. Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.

    • Michal Domanski
    •  & Torben Heick Jensen
  3. Orochem Technologies Inc., Naperville, Illinois, USA.

    • Asha A Oroskar
    •  & Anil R Oroskar
  4. Center for Health Informatics and Bioinformatics, New York University School of Medicine, New York, New York, USA.

    • Sarah Keegan
    •  & David Fenyö
  5. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, USA.

    • Sarah Keegan
    •  & David Fenyö
  6. Institute for Systems Biology, Seattle, Washington, USA.

    • David J Dilworth
    •  & John D Aitchison
  7. Seattle Biomedical Research Institute, Seattle, Washington, USA.

    • David J Dilworth
    •  & John D Aitchison
  8. Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York, USA.

    • Kelly R Molloy
    •  & Brian T Chait
  9. High Energy Physics Instrument Shop, The Rockefeller University, New York, New York, USA.

    • Vadim Sherman

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Contributions

J.L. and M.P.R. conceived the screening strategy; J.L. carried out proof-of-concept experiments, assisted by L.E.H.; L.E.H. and V.S. designed manifolds, which were fabricated by V.S. and tested by L.E.H., J.L. and Z.H.; filters were designed by A.A.O., A.R.O. and J.L., fabricated by A.A.O. and A.R.O., and tested by J.L. and Z.H.; J.L., Z.H. and M.D. designed experiments, executed screens and further developed procedures—with yeast work primarily carried out by Z.H. and human cell line work primarily carried out by M.D.; MS analyses were carried out by J.L., Z.H. and K.R.M., with I-DIRT done by Z.H.; transposing the procedure to robotic automation was carried out by D.J.D. assisted by J.L.; J.L., Z.H. and D.F. conceived of the protein copurification gel database and software, which was built by S.K. and D.F. with testing and feedback from J.L. and Z.H.; J.D.A., D.F., B.T.C., T.H.J., M.P.R. and J.L. supervised the project; Z.H., B.T.C., M.P.R. and J.L. wrote the paper.

Competing interests

A.A.O., A.R.O., M.P.R. and J.L. are inventors on a US patent application encompassing the filter work described in this article.

Corresponding authors

Correspondence to Michael P Rout or John LaCava.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15, Supplementary Tables 1, 3 and 5, Supplementary Notes 1 and 2, Supplementary Data and Supplementary Protocol 1

Excel files

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    Supplementary Table 2

    LC-MS/MS data of Nup1p-SpA affinity capture

  2. 2.

    Supplementary Table 4

    MS data for Rtn1p affinity capture experiments

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    Supplementary Protocol 2

    Program files for Hamilton STAR liquid handling workstation

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DOI

https://doi.org/10.1038/nmeth.3395

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