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

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

Author information

1
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093-0378, USA.
2
Proteomic Service Group Cell Signaling Technology, 3 Trask Lane, Danvers, MA, 01923, USA.
3
Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, 92093-0380, USA.
4
Broad Institute of MIT and Harvard, 415 Main St, Cambridge, MA, 02142, USA.
5
Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, 92093-0380, USA. e1bennett@ucsd.edu.
6
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093-0378, USA. ekomives@ucsd.edu.

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.

PMID:
29079793
PMCID:
PMC5660086
DOI:
10.1038/s41467-017-01271-3
[Indexed for MEDLINE]
Free PMC Article

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