An improved SUMmOn-based methodology for the identification of ubiquitin and ubiquitin-like protein conjugation sites identifies novel ubiquitin-like protein chain linkages.
Ubiquitin (Ub) and the ubiquitin-like proteins (Ubls) comprise a remarkable assortment of polypeptides that are covalently conjugated to target proteins (or other biomolecules) to modulate their intracellular localization, half-life, and/or activity. Identification of Ub/Ubl conjugation sites on a protein of interest can thus be extremely important for understanding how it is regulated. While MS has become a powerful tool for the study of many classes of PTMs, the identification of Ub/Ubl conjugation sites presents a number of unique challenges. Here, we present an improved Ub/Ubl conjugation site identification strategy, utilizing SUMmOn analysis and an additional protease (lysyl endopeptidase C), as a complement to standard approaches. As compared with standard trypsin proteolysis-database search protocols alone, the addition of SUMmOn analysis can (i) identify Ubl conjugation sites that are not detected by standard database searching methods, (ii) better preserve Ub/Ubl conjugate identity, and (iii) increase the number of identifications of Ub/Ubl modifications in lysine-rich protein regions. Using this methodology, we characterize for the first time a number of novel Ubl linkages and conjugation sites, including alternative yeast (K54) and mammalian small ubiquitin-related modifier (SUMO) chain (SUMO-2 K42, SUMO-3 K41) assemblies, as well as previously unreported NEDD8 chain (K27, K33, and K54) topologies.
Jeram
Stanley M
SM
Ontario Cancer Institute, and McLaughlin Centre for Molecular Medicine, Toronto, ON, M5G 1L7, Canada.
Srikumar
Tharan
T
Zhang
Xiang-Dong
XD
Anne Eisenhauer
H
H
Rogers
Richard
R
Pedrioli
Patrick G A
PG
Matunis
Michael
M
Raught
Brian
B
D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D016415:Sequence Alignment; D025841:Small Ubiquitin-Related Modifier Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin