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

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...

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Bibliographic Details
Published in:Nature communications 2017-10, Vol.8 (1), p.1171-11, Article 1171
Main Authors: Lumpkin, Ryan J., Gu, Hongbo, Zhu, Yiying, Leonard, Marilyn, Ahmad, Alla S., Clauser, Karl R., Meyer, Jesse G., Bennett, Eric J., Komives, Elizabeth A.
Format: Article
Language:English
Subjects:
631/337/458/538
631/337/475/2290
Ectopic expression
Humanities and Social Sciences
Lysine
Mass spectrometry
Mass spectroscopy
multidisciplinary
Peptides
Post-translation
Protease
Proteasomes
Proteinase
Proteins
Proteomes
Quantitation
Science
Science (multidisciplinary)
SUMO protein
Ubiquitin
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Summary: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. SUMOylation is post-translational modification implicated in several biological pathways. Here the authors describe an approach for the global profiling of SUMO attachment sites under native conditions that also allows the parallel determination of SUMO and Ub attachments.
ISSN:2041-1723
2041-1723
DOI:10.1038/s41467-017-01271-3