Proteome-wide identification of SUMO modification sites by mass spectrometry.
The protein called 'small ubiquitin-like modifier' (SUMO) is post-translationally linked to target proteins at the ɛ-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-ɛ-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.
Centre for Gene Regulation and Expression, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK.
Triin
T
Tammsalu
Centre for Gene Regulation and Expression, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK.
Ivan
I
Matic
Centre for Gene Regulation and Expression, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK.
Ellis G
EG
Jaffray
MRC Protein Phosphorylation and Ubiquitination Unit, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK.
Adel F M
AF
Ibrahim
Centre for Gene Regulation and Expression, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK.
Michael H
MH
Tatham
Centre for Gene Regulation and Expression, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, UK.
Ronald T
http://orcid.org/0000-0001-7113-9024
RT
Hay
D000595:Amino Acid Sequence; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods*; D058207:Sumoylation