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Abstract
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 language | English |
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Pages (from-to) | 1374-1388 |
Number of pages | 15 |
Journal | Nature Protocols |
Volume | 10 |
Issue number | 9 |
DOIs | |
Publication status | Published - Sept 2015 |
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
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Dive into the research topics of 'Proteome-wide identification of SUMO modification sites by mass spectrometry'. Together they form a unique fingerprint.Projects
- 2 Finished
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Strategic Award: Wellcome Trust Technology Platform
Blow, J., Lamond, A. & Owen-Hughes, T.
1/01/13 → 30/09/18
Project: Research
Student theses
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Global identification of SUMO modification sites
Author: Tammsalu, T., 2016Supervisor: Hay, R. (Supervisor)
Student thesis: Doctoral Thesis › Doctor of Philosophy
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