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

Nat Commun. 2017 Oct 27;8(1):1171. doi: 10.1038/s41467-017-01271-3.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • HeLa Cells
  • Humans
  • Lysine / chemistry
  • Mass Spectrometry
  • Mutation
  • Peptides / chemistry
  • Protein Binding
  • Protein Domains
  • Protein Processing, Post-Translational
  • Proteome
  • Proteomics
  • Serine Endopeptidases / chemistry*
  • Signal Transduction
  • Small Ubiquitin-Related Modifier Proteins / metabolism*
  • Sumoylation
  • Trypsin / chemistry
  • Ubiquitin / chemistry


  • Peptides
  • Proteome
  • Small Ubiquitin-Related Modifier Proteins
  • Ubiquitin
  • Serine Endopeptidases
  • myxobacter alpha-lytic proteinase
  • Trypsin
  • Lysine