Proteome-wide identification of SUMO modification sites by mass spectrometry

The protein called 'small ubiquitin-like modifier/(SUSUMO) is post-translationally linked to target proteins at the. -amino group of lysine residues. This 'SUSUMOylation/alters the behavior of the target protein, a change that is utilized to regulate diverse cellular processes. Understanding the target-specific consequences of SUSUMO modification requires knowledge of the location of conjugation sites, and we have developed a straightforward protocol for the proteome-wide identification of SUSUMO modification sites using mass spectrometry (MS). The approach described herein requires the expression of a mutant form of SUSUMO, in which the residue preceding the C-terminal Gly-Gly (diGly) is replaced with a lysine (SUSUMOKGG). Digestion of SUSUMOKGG 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-epsilon-GG)-specific antibody and identified by MS. This diGly signature is characteristic of SUSUMOKGG 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 SUSUMOylation 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 similar to 5 d.