Synergistic Arg-C Ultra and Lys-C Digestion for Quantitative Proteomics.

ABSTRACT: Shotgun proteomics hinges on complete enzymatic digestion of proteins into peptides. Incomplete digestion narrows proteome coverage and inflates variability in quantitative workflows, whether label-free DIA or multiplexing with isobaric tags. Sequential Lys-C/Trypsin digestions mitigate missed cleavages at lysine residues, but arginine sites remain a persistent challenge. Arg-C Ultra, a recently released cysteine protease, efficiently targets arginine residues but requires reducing conditions that inactivate Lys-C activity and compromise NHS-ester labeling in multiplexed workflows. Here, we systematically characterized Arg-C Ultra and Lys-C with chromogenic substrates that mimic arginine- and lysine-containing peptides, as well as shotgun proteomics. Arg-C Ultra operates optimally at room

SUBMITTER: Pujari V 

PROVIDER: S-EPMC12338676 | biostudies-literature | 2025 Jul

REPOSITORIES: biostudies-literature