An improved SUMmOn-based methodology for the identification of ubiquitin and ubiquitin-like protein (Ubl) conjugation sites identifies novel Ubl chain linkages

摘要

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 mass spectrometry (MS) has become a powerful tool for the study of many classes of post-translational modifications, 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 (LysC), as a complement to standard approaches. As compared to standard trypsin proteolysis-database search protocols alone, the addition of SUMmOn analysis can; (a) identify Ubl conjugation sites that are not detected by standard database searching methods, (b) better preserve Ub/Ubl conjugate identity, and (c) 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 SUMO chain (SUMO-2 K42, SUMO-3 K41) assemblies, as well as previously unreported NEDD8 chain (K27, K33 and K54) topologies.