Proteome-wide identification of SUMO modification sites by mass spectrometry

Triin Tammsalu, Ivan Matic, Ellis G. Jaffray, Adel F. M. Ibrahim, Michael H. Tatham, Ronald T. Hay (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

45 Citations (Scopus)


The protein called 'small ubiquitin-like modifier' (SUMO) is post-translationally linked to target proteins at the η -amino group of lysine residues. This 'SUMOylation' alters the behavior of the target protein, a change that is utilized to regulate diverse cellular processes. Understanding the target-specific consequences of SUMO modification requires knowledge of the location of conjugation sites, and we have developed a straightforward protocol for the proteome-wide identification of SUMO modification sites using mass spectrometry (MS). The approach described herein requires the expression of a mutant form of SUMO, in which the residue preceding the C-terminal Gly-Gly (diGly) is replaced with a lysine (SUMO KGG). Digestion of SUMO KGG protein conjugates with endoproteinase Lys-C yields a diGly motif attached to target lysines. Peptides containing this adduct are enriched using a diGly-Lys (K-η -GG)-specific antibody and identified by MS. This diGly signature is characteristic of SUMO KGG conjugation alone, as no other ubiquitin-like protein (Ubl) yields this adduct upon Lys-C digestion. We have demonstrated the utility of the approach in SUMOylation studies, but, in principle, it may be adapted for the site-specific identification of proteins modified by any Ubl. Starting from cell lysis, this protocol can be completed in ∼5 d.

Original languageEnglish
Pages (from-to)1374-1388
Number of pages15
JournalNature Protocols
Issue number9
Publication statusPublished - Sept 2015

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)


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