Ac-LysargiNase Complements Trypsin for the Identification of Ubiquitinated Sites

Mass spectrometry (MS)-based identification of ubiquitinated sites
requires trypsin digestion prior to MS analysis, and a signature peptide
was produced with a diglycine residue attached to the ubiquitinated
lysine (K-ε-GG peptide). However, the missed cleavage of modified
lysines by trypsin results in modified peptides with increased length
and charge, whose detection by MS analysis is suppressed by the vast
majority of internally unmodified peptides. LysargiNase, the mirrored
trypsin, is reported to cleave before lysine and arginine residues
and to be favorable for the identification of methylation and phosphorylation,
but its digestive characteristics related to ubiquitination are unclear.
Herein, we tested the capacity of the in-house developed acetylated
LysargiNase (Ac-LysargiNase) with high activity and stability, for
cleaving ubiquitinated sites in both the seven types of ubiquitin
chains and their corresponding K-ε-GG peptides. Interestingly,
Ac-LysargiNase could efficiently cleave the K63-linked chain but had
little effect on the other types of chains. Additionally, Ac-LysargiNase
had higher exopeptidase activity than trypsin. Utilizing these features
of the paired mirror proteases, a workflow of trypsin and Ac-LysargiNase
tandem digestion was developed for the identification of ubiquitinated
proteins. Through this method, the charge states and ionization capacity
of the unmodified peptides were efficiently reduced, and the identification
of modified sites was consequently increased by 30% to 50%. Strikingly,
approximately 15% of the modified sites were cleaved by Ac-LysargiNase,
resulting in shorter K-ε-GG peptides for better identification.
The enzyme Ac-LysargiNase is expected to serve as an option for increasing
the efficiency of modified site identification in ubiquitome research.