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Reference: Jeram SM, et al. (2010) An improved SUMmOn-based methodology for the identification of ubiquitin and ubiquitin-like protein conjugation sites identifies novel ubiquitin-like protein chain linkages. Proteomics 10(2):254-65

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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.

Reference Type
Journal Article
Authors
Jeram SM, Srikumar T, Zhang XD, Anne Eisenhauer H, Rogers R, Pedrioli PG, Matunis M, Raught B
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