The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His 6 tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We The small ubiquitin-like modifier (SUMO) 1 proteins are structurally similar to ubiquitin, although they share less than 20% sequence identity (1). Like ubiquitylation, protein SUMOylation is regulated by a cascade of reactions involving SUMO-activating enzymes (SAE1/SAE2), -conjugating enzymes (Ubc9), and one of several SUMO E3 ligases (e.g. PIAS1, PIAS3, PIASx␣, PIASx, PIASy, RanBP2, and Pc2) that covalently attach SUMO to specific protein substrates (2, 3). SUMO proteins are expressed as an immature proform that comprises an invariant Gly-Gly motif followed by a Cterminal stretch of variable length (2-11 amino acids). Removal of this C-terminal extension by sentrin-specific proteases (SENPs) to expose the diglycine motif is necessary for the conjugation of SUMO to protein targets. These SUMO proteases are able to cleave both a peptide bond during the formation of mature SUMO and an isopeptide bond to deconjugate modified protein substrates (4). This covalent modification arises from the formation of an isopeptide bond between the -amino group of a lysine within the protein substrate and the C terminus carboxyl group of the SUMO glycine residue. SUMO conjugation frequently occurs at the lysine residue within the consensus motif KXE (where is an aliphatic residue and X is any amino acid) that is recognized by Ubc9 (5, 6). Recent studies have also identified a phosphorylation-dependent motif (⌿KXEXXpSP where pS is phosphoserine) (7) and a negatively charged amino acid-dependent motif (8) that harbor negative charges next to the basic SUMO consensus site to enhance protein SUMOylation. However, several other SUMOylated proteins including proliferating cell nuclear antigen, E2-25K, Daxx (death domainassociated protein), and USP25 are modified at non-consensus sites (9 -11). Whether these types of sites are rare 1 The abbreviations used are: SUMO, small ubiquitin-related modifier; As 2 O 3 , arsenic trioxide; E2-25K, E2 ubiquitin ligase, 25 kilodalto...