title: An improved SUMmOn-based methodology for the identification of ubiquitin and ubiquitin-like protein conjugation sites identifies novel ubiquitin-like protein chain linkages.
abstract: 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.
authors.affiliation: Ontario Cancer Institute, and McLaughlin Centre for Molecular Medicine, Toronto, ON, M5G 1L7, Canada.
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Jeram
authors.forename: Tharan
authors.initials: T
authors.lastname: Srikumar
authors.forename: Xiang-Dong
authors.initials: XD
authors.lastname: Zhang
authors.forename: H
authors.initials: H
authors.lastname: Anne Eisenhauer
authors.forename: Richard
authors.initials: R
authors.lastname: Rogers
authors.forename: Patrick G A
authors.initials: PG
authors.lastname: Pedrioli
authors.forename: Michael
authors.initials: M
authors.lastname: Matunis
authors.forename: Brian
authors.initials: B
authors.lastname: Raught
mesh_terms: 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
chemical_list: D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin