Abstract

Efforts to map the human protein interactome have resulted in information about thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we report a computational search strategy that supports hierarchical top-down analysis for precise identification and scoring of multi-proteoform complexes by native mass spectrometry.

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Acknowledgements

The authors thank members of the Kelleher research group and V. Wysocki for helpful discussions and advice. O.S.S. is supported by a US National Science Foundation Graduate Research Fellowship (2014171659). P.C.H. is a recipient of a Northwestern University Chemistry of Life Processes Institute Postdoctoral Fellowship Award. L.H.F.D.V. is supported under CNPq research grant 202011/2012-7 from the Brazilian government. H.S.S. is supported under the Science Without Borders scholarship 88888.075416/2013-00 from the Coordination for the Improvement of Higher Education Personnel under the Brazilian government. This work was supported by grants from the W.M. Keck Foundation (DT061512) and the US National Institutes of Health (GM067193) to N.L.K.

Author information

Author notes

    • Owen S Skinner
    •  & Pierre C Havugimana

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemistry, Northwestern University, Evanston, Illinois, USA.

    • Owen S Skinner
    • , Nicole A Haverland
    • , Luca Fornelli
    • , Rafael D Melani
    • , Henrique S Seckler
    • , Philip D Compton
    •  & Neil L Kelleher
  2. Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois, USA.

    • Pierre C Havugimana
    • , Luca Fornelli
    • , Richard D LeDuc
    •  & Neil L Kelleher
  3. Proteomics Center of Excellence, Northwestern University, Evanston, Illinois, USA.

    • Pierre C Havugimana
    • , Luca Fornelli
    • , Bryan P Early
    • , Joseph B Greer
    • , Ryan T Fellers
    • , Luis H F Do Vale
    • , Philip D Compton
    •  & Neil L Kelleher
  4. Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA.

    • Kenneth R Durbin
    •  & Neil L Kelleher
  5. Brazilian Center for Protein Research, University of Brasilia, Brasilia, Federal District, Brazil.

    • Luis H F Do Vale
  6. Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona, USA.

    • Micah T Nelp
    •  & Vahe Bandarian
  7. Thermo Fisher Scientific (Bremen) GmbH, Bremen, Germany.

    • Mikhail E Belov
    • , Stevan R Horning
    •  & Alexander A Makarov

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Contributions

P.C.H. and N.L.K. devised the computational approach with contributions from O.S.S., R.D.L. and R.T.F. O.S.S. interpreted the data with help from P.C.H. and P.D.C. O.S.S. and P.D.C. collected native MS data. Further supporting results were collected by L.F., K.R.D., L.H.F.D.V. and R.D.M. R.D.L. developed the MPC score. B.P.E., J.B.G. and R.T.F. implemented the search engine and scoring system. M.E.B., S.R.H. and A.A.M. participated in the design of the instrument and support of initial experiments. M.T.N. and V.B. provided the TNH complex. O.S.S., P.C.H. and N.L.K. co-wrote the manuscript with critical inputs from N.A.H. and H.S.S. and contributions from all coauthors. N.L.K. initiated and supervised the project.

Competing interests

M.E.B., S.R.H. and A.A.M. are employees of Thermo Fisher Scientific, and R.D.L., N.L.K., R.T.F. and J.B.G. help commercialize ProSightPC.

Corresponding author

Correspondence to Neil L Kelleher.

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    Supplementary Figures 1–8, Supplementary Discussion and Supplementary Tables 1–4.

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    Neutral monoisotopic masses for use with the web tool

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DOI

https://doi.org/10.1038/nmeth.3731

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