UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites

Ubiquitinationis a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitinationat a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitinationon lysine residues, protein N-terminal ubiquitinationis readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitinationsites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitinationand acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitinationsites upon proteasome inhibition.