A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program.
Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described.
Knuesel
Matthew
M
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
Cheung
Hiu Tom
HT
Hamady
Micah
M
Barthel
Kristen K B
KK
Liu
Xuedong
X
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics; D019295:Computational Biology / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D020690:GTPase-Activating Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D020407:Internet; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D010449:Peptide Mapping; D011817:Rabbits; D012156:Reticulocytes / Q000166:cytology; D025842:SUMO-1 Protein / Q000032:analysis / Q000378:metabolism*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012984:Software; D013997:Time Factors; D025801:Ubiquitin / Q000737:chemistry; D014584:User-Computer Interface
D018076:DNA, Complementary; D020690:GTPase-Activating Proteins; C085844:Rangap1 protein, mouse; D025842:SUMO-1 Protein; D025801:Ubiquitin; D001120:Arginine