Proteome-wide identification of SUMO2 modification sites

Posttranslational modification with small ubiquitin-like modifiers (SUMOs) alters the function of proteins involved in diverse cellular processes. SUMOs are conjugated to lysine residues in target proteins by SUMO-specific enzymes. Although proteomic studies have identified hundreds of sumoylated substrates, methods to identify the modified lysines on a proteome-wide scale are lacking. We developed a method that enabled large-scale identification of sumoylated lysines and involved the expression of polyhistidine (6His)–tagged SUMO2 with Thr90 mutated to Lys. Digestion of 6His-SUMO2(T90K)–modified proteins with an endoproteinase Lys-C produces a diGly remnant on SUMO2(T90K)-conjugated lysines, enabling a specific immunoprecipitation of modified peptides with diGly-Lys-specific antibody and producing a unique mass-to-charge signature. Mass spectrometry analysis of SUMO2-enriched peptides from human cell lysates revealed more than 1000 sumoylated lysines in 539 proteins, including many functionally related proteins involved in cell cycle, transcription, and DNA repair. Not only can this strategy be used to study the dynamics of sumoylation and other potentially similar posttranslational modifications, but also, these data provide an unprecedented resource for future research on the role of sumoylation in cellular physiology and disease.