Proteome-wide identification of SUMO modification sites by mass spectrometry

Abstract

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.

A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

“…Twinstar (Tsr) is the Drosophila orthologue of the actin destabilizing factor cofilin. Previous work has demonstrated SUMOylation of human cofilin4344. Interestingly, we note that one of the identified SUMOylation sites of human cofilin (K13) overlaps with its reported nuclear export sequence (VIKVFNDMKV)45, which might have important consequences on localization for a small protein like Tsr (17 kDa).…”

A SUMO-Dependent Protein Network Regulates Chromosome Congression during Oocyte Meiosis

“…To search for SUMO substrates during meiosis, we adapted to C. elegans a proteomics approach successfully employed in human cells (Tammsalu et al., 2014, Tammsalu et al., 2015). To identify sumoylation sites in vivo, we generated worms expressing His 6 -tagged SUMO with a Leu to Lys substitution preceding the C-terminal diGly motif (L88K) in the germline and early embryos (Figure S3A).…”

“…To identify sumoylation sites in vivo, we generated worms expressing His 6 -tagged SUMO with a Leu to Lys substitution preceding the C-terminal diGly motif (L88K) in the germline and early embryos (Figure S3A). After Ni-NTA purification and Lys-C digestion, the mutant SUMO leaves a GG remnant on substrate lysines that facilitates peptide enrichment with an anti-K-ε-GG antibody (Figure S3B) (Tammsalu et al., 2014, Tammsalu et al., 2015). Conjugation of SUMO(L88K) to substrates in vitro is indistinguishable from wild-type SUMO (Figures S3C and S3D) and is also conjugated in vivo (Figure S3E).…”

The Roles of SUMO in Metabolic Regulation

“…The number of identified Sumoylated proteins, and proteins that interact with SUMO through SUMO interacting motifs (SIM), continues to grow (Becker et al 2013; Bruderer et al 2011; Eifler and Vertegaal 2015; Hendriks et al 2014; Impens et al 2014; Jardin et al 2015; Jentsch and Psakhye 2013; Kaminsky et al 2009; Kroetz and Hochstrasser 2009; Lamoliatte et al 2014; Makhnevych et al 2009; Subramonian et al 2014; Tammsalu et al 2014, 2015; Yang and Paschen 2015; Yang et al 2012). Like other forms of post-translational modification, sumoylation is now known to be involved in most, if not all cellular processes (Flotho and Melchior 2013; Gareau and Lima 2010; Hecker et al 2006; Makhnevych et al 2009; Stehmeier and Muller 2009; Wilkinson and Henley 2010).…”