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Site-specific identification and quantitation of endogenous SUMO modifications under native conditions

Author

Listed:
  • Ryan J. Lumpkin

    (Department of Chemistry and Biochemistry, University of California, San Diego)

  • Hongbo Gu

    (Proteomic Service Group Cell Signaling Technology)

  • Yiying Zhu

    (Proteomic Service Group Cell Signaling Technology)

  • Marilyn Leonard

    (University of California, San Diego)

  • Alla S. Ahmad

    (Department of Chemistry and Biochemistry, University of California, San Diego)

  • Karl R. Clauser

    (Broad Institute of MIT and Harvard)

  • Jesse G. Meyer

    (Department of Chemistry and Biochemistry, University of California, San Diego)

  • Eric J. Bennett

    (University of California, San Diego)

  • Elizabeth A. Komives

    (Department of Chemistry and Biochemistry, University of California, San Diego)

Abstract

Small ubiquitin-like modifier (SUMO) modification regulates numerous cellular processes. Unlike ubiquitin, detection of endogenous SUMOylated proteins is limited by the lack of naturally occurring protease sites in the C-terminal tail of SUMO proteins. Proteome-wide detection of SUMOylation sites on target proteins typically requires ectopic expression of mutant SUMOs with introduced tryptic sites. Here, we report a method for proteome-wide, site-level detection of endogenous SUMOylation that uses α-lytic protease, WaLP. WaLP digestion of SUMOylated proteins generates peptides containing SUMO-remnant diglycyl-lysine (KGG) at the site of SUMO modification. Using previously developed immuno-affinity isolation of KGG-containing peptides followed by mass spectrometry, we identified 1209 unique endogenous SUMO modification sites. We also demonstrate the impact of proteasome inhibition on ubiquitin and SUMO-modified proteomes using parallel quantitation of ubiquitylated and SUMOylated peptides. This methodological advancement enables determination of endogenous SUMOylated proteins under completely native conditions.

Suggested Citation

  • Ryan J. Lumpkin & Hongbo Gu & Yiying Zhu & Marilyn Leonard & Alla S. Ahmad & Karl R. Clauser & Jesse G. Meyer & Eric J. Bennett & Elizabeth A. Komives, 2017. "Site-specific identification and quantitation of endogenous SUMO modifications under native conditions," Nature Communications, Nature, vol. 8(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_s41467-017-01271-3
    DOI: 10.1038/s41467-017-01271-3
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