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. SUMO-specific enzymes conjugate SUMOs to lysine residues in target proteins. Although proteomic studies have identified hundreds of sumoylated substrates, methods to identify the modified lysines on a proteomic scale are lacking. We developed a method that enabled proteome-wide identification of sumoylated lysines that involves the expression of polyhistidine (6His)-tagged SUMO2 with Thr(90) mutated to Lys. Endoproteinase cleavage with Lys-C of 6His-SUMO2(T90K)-modified proteins from human cell lysates produced a diGly remnant on SUMO2(T90K)-conjugated lysines, enabling immunoprecipitation of SUMO2(T90K)-modified peptides and producing a unique mass-to-charge signature. Mass spectrometry analysis of SUMO-enriched peptides 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.
Centre for Gene Regulation and Expression, University of Dundee, Sir James Black Centre, Dow Street, Dundee DD1 5EH. UK.
Triin
T
Tammsalu
Centre for Gene Regulation and Expression, University of Dundee, Sir James Black Centre, Dow Street, Dundee DD1 5EH. UK.
Ivan
I
Matic
Centre for Gene Regulation and Expression, University of Dundee, Sir James Black Centre, Dow Street, Dundee DD1 5EH. UK.
Ellis G
EG
Jaffray
MRC Protein Phosphorylation and Ubiquitination Unit, College of Life Sciences, University of Dundee, Sir James Black Centre, Dow Street, Dundee DD1 5EH. UK.
Adel F M
AFM
Ibrahim
Centre for Gene Regulation and Expression, University of Dundee, Sir James Black Centre, Dow Street, Dundee DD1 5EH. UK.
Michael H
MH
Tatham
Centre for Gene Regulation and Expression, University of Dundee, Sir James Black Centre, Dow Street, Dundee DD1 5EH. UK.
Ronald T
RT
Hay
D000595:Amino Acid Sequence; D015153:Blotting, Western; D057809:HEK293 Cells; D006639:Histidine / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D010455:Peptides; D020543:Proteome; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; C033223:polyhistidine; D013912:Threonine; D006639:Histidine; D008239:Lysine