Abstract

Cellular function is critically regulated through degradation of substrates by the proteasome. To enable direct analysis of naturally cleaved proteasomal peptides under physiological conditions, we developed mass spectrometry analysis of proteolytic peptides (MAPP), a method for proteasomal footprinting that allows for capture, isolation and analysis of proteasome-cleaved peptides. Application of MAPP to cancer cell lines as well as primary immune cells revealed dynamic modulation of the cellular degradome in response to various stimuli, such as proinflammatory signals. Further, we performed analysis of minute amounts of clinical samples by studying cells from the peripheral blood of patients with systemic lupus erythematosus (SLE). We found increased degradation of histones in patient immune cells, thereby suggesting a role of aberrant proteasomal degradation in the pathophysiology of SLE. Thus, MAPP offers a broadly applicable method to facilitate the study of the cellular-degradation landscape in various cellular conditions and diseases involving changes in proteasomal degradation, including protein aggregation diseases, autoimmunity and cancer.

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Acknowledgements

We thank K. Tanaka and S. Murata (University of Tokyo) for the anti-PSMA1 hybridoma, and J. Bar and I. Kamer (Sheba Medical Center) for the anti-PD-1 monoclonal antibody Nivolumab. We thank members of the Merbl laboratory and M. Sharon for critically reviewing the manuscript; S. Itzkovitz, M. Askenazi, S. Shen-Orr, I. Amit and E. Elinav for scientific advice; and M. Kupervaser and A. Savidor for analytic support. This project received funding from Merck KGaA; the European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation Programme (grant agreement 677748); the I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation (grant 12/7751); Rothschild Caesarea; The Peter & Patricia Gruber Foundation (Gruber award); Adiel Eleanor, Carla Hunter and Andre Schub (Y.M.); and the Intramural Research Program at NIAMS/NIH ZIA AR041199 (C.C.-R. and M.J.K.). Y.M. is supported as the incumbent Leonard and Carol Berall Career Development Chair.

Author information

Author notes

    • Hila Wolf-Levy
    • , Aaron Javitt
    •  & Avital Eisenberg-Lerner

    These authors contributed equally to this work.

Affiliations

  1. Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

    • Hila Wolf-Levy
    • , Aaron Javitt
    • , Avital Eisenberg-Lerner
    • , Assaf Kacen
    • , Adi Ulman
    • , Daoud Sheban
    • , Bareket Dassa
    • , Vered Fishbain-Yoskovitz
    • , Matthias P Kramer
    • , Neta Nudel
    • , Ifat Regev
    • , Liron Zahavi
    •  & Yifat Merbl
  2. Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health (NIH), Bethesda, Maryland, USA.

    • Carmelo Carmona-Rivera
    •  & Mariana J Kaplan
  3. De Botton Institute for Protein Profiling, Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot, Israel.

    • Dalia Elinger
    • , David Morgenstern
    •  & Yishai Levin

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Contributions

Y.M., H.W.-L., A.E.-L., A.U., D.S., M.P.K., N.N. and I.R. designed and performed experiments and interpreted the data. Y.M., A.J., H.W.-L., A.K., B.D., V.F.-Y. and L.Z. performed and interpreted computational analyses. Y.M., A.E.-L. and A.J. wrote the manuscript. H.W.-L., D.E., D.M. and Y.L. performed MS analysis. C.C.-R. and M.J.K. administered the human SLE research and provided clinical advice. Y.M. conceived the concept and oversaw the study.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Avital Eisenberg-Lerner or Yifat Merbl.

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Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–11

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Note

    Extended Mass Spectrometry Methods

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    Supplementary Data 1

    26S proteasome subunits identified in MAPP eluate.

  2. 2.

    Supplementary Data 2

    Peptides identified by MAPP of cells stimulated with TNFα and IFNγ or left untreated

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    Supplementary Data 3

    Proteins with increased degradation and unchanged abundance.

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    Supplementary Data 4

    Proteins clustered by degradation trend

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    Supplementary Data 5

    PD-1 blockade changed MAPP intensity by 2-fold or more in 168 proteins

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    Supplementary Data 6

    45 components of the ubiquitin system

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    Supplementary Data 7

    Patient information

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    Supplementary Data 8

    Peptides identified in MAPP of SLE patients and healthy individuals

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DOI

https://doi.org/10.1038/nbt.4279