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Journal Article

Proteome-wide identification of SUMO modification sites by mass spectrometry


Matić,  I.
Matic – ADP-ribosylation in DNA Repair and Ageing, Research Groups, Max Planck Institute for Biology of Ageing, Max Planck Society;

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Tammsalu, T., Matić, I., Jaffray, E. G., Ibrahim, A. F., Tatham, M. H., & Hay, R. T. (2015). Proteome-wide identification of SUMO modification sites by mass spectrometry. Nat Protoc, 10(9), 1374-88. doi:10.1038/nprot.2015.095.

Cite as: https://hdl.handle.net/21.11116/0000-000B-6E73-9
The protein called 'small ubiquitin-like modifier' (SUMO) is post-translationally linked to target proteins at the varepsilon-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-varepsilon-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 approximately 5 d.