Large-Scale Profiling of Unexpected Tryptic Cleaved Sites at Ubiquitinated Lysines

Trypsin specifically cleaves the C-terminus of lysine and arginine residues but often fails to cleave modified lysines, such as ubiquitination, therefore resulting in the uncleaved K-e-GG peptides. Therefore, the cleaved ubiquitinated peptide identification was often regarded as false positives and discarded. Interestingly, unexpected cleavage at the K48-linked ubiquitin chain has been reported, suggesting the latent ability of trypsin to cleave ubiquitinated lysine residues. However, it remains unclear whether other trypsin-cleavable ubiquitinated sites are present. In this study, we verified the ability of trypsin in cleaving K6 and K63 besides K48 chains. The uncleaved K-e-GG peptide was quickly and efficiently generated during trypsin digestion, whereas cleaved ones were produced with much lower efficiency. Then, the K-e- GG antibody was proved to efficiently enrich the cleaved K-e-GG peptides and several published large-scale ubiquitylation datasets were re-analyzed to interrogate the cleaved sequence features. In total, more than 2400 cleaved ubiquitinated peptides were identified in the K-e-GG and UbiSite antibody-based datasets. The frequency of lysine upstream of the cleaved modified K was significantly enriched. The kinetic activity of trypsin in cleaving ubiquitinated peptides was further elucidated. We suggest that the cleaved K-e-GG sites with high post-translational modification probability (>= 0.75) should be considered as true positives in future ubiquitome analyses.

Automated summary

Trypsin is known for cleaving the C-terminus of lysine and arginine residues, but it often fails to cleave modified lysines, such as ubiquitination. This leads to the presence of uncleaved K-e-GG peptides, which are usually discarded as false positives. In this study, the ability of trypsin to cleave other ubiquitinated sites besides K48 chains was verified. A significant enrichment of lysine upstream of the cleaved modified K was observed. The findings suggest that cleaved K-e-GG sites with high post-translational modification probability (>=0.75) should be considered as true positives in future ubiquitome analyses.