Nat. Struct. Mol. Biol. 25, 631–640 (2018)

An antibody recognizing lysine-diGly-containing peptides formed during trypsin digestion of ubiquitinated proteins is commonly used to enrich ubiquitinated peptides for mass spectrometry analysis of ubiquitination sites. However, N-terminal ubiquitination cannot be detected with this antibody, and the two ubiquitin-like modifiers NEDD8 and ISG15 also yield lysine-diGly peptides after tryptic digestion, which limits the specificity of this approach. Akimov et al. overcame these limitations by raising a monoclonal antibody, UbiSite, which is specific for a 13-residue peptide formed during the digestion of ubiquitinated proteins with endoproteinase LysC. In the UbiSite workflow, proteins extracted from cultured cells or tissues are digested with LysC, enriched with the UbiSite antibody and analyzed by LC–MS/MS either directly or after additional trypsin treatment. The authors analyzed the effect of proteasome inhibitors on the ubiquitinomes of two human cell lines and identified more than 63,000 unique ubiquitination sites in over 9,200 proteins. Furthermore, N-terminal ubiquitination of more than 100 proteins was detected, which was also verified by mutagenesis studies. UbiSite improves the specificity of ubiquitinome analysis and might also be a useful tool to study the function and regulation of N-terminal ubiquitination.