@article{204451,
  author = {Vyas Pujari and Joseph Crapse and Connor Nisbet and Gloria Bao and Wessley Ferguson and Christopher Hosfield and Michael Rosenblatt and Felix Keber and Martin W{\"u}hr},
  title = {Synergistic Arg-C Ultra and Lys-C Digestion for Quantitative Proteomics},
  abstract = {<p>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 temperature, pH 7.5{\textendash}8.5, under reducing conditions, whereas Lys-C is most active at 37 {\textdegree}C, pH 7.5{\textendash}8.5, yet rapidly loses activity when exposed to common reductants. Among tested reducing agents, 1 mM TCEP uniquely preserved TMTpro integrity while sustaining Arg-C Ultra activity. Guided by these insights, we established a sequential digestion workflow that is fully compatible with both label- free DIA and TMTpro multiplexing. Proteins are first digested overnight with Lys-C at 37 {\textdegree}C (pH 8.5), then treated with 1 mM TCEP and Arg-C Ultra at room temperature (pH 8.5). The resulting peptides can be analyzed directly by label-free DIA or subjected to TMTpro labeling for multiplexed quantification. Applied to HeLa cell lysates, this protocol achieved \&gt;99\% arginine and 95\% lysine cleavage efficiencies, boosting the number of quantified proteins by 6\% in label- free DIA and 11\% in TMTproC experiments. Replicate measurements displayed reproducibility that approached the limits set by ion statistics. Thus, the introduced synergistic Lys-C/Arg-C Ultra digestion strategy enhances proteome coverage with excellent quantitative reproducibility across both label-free and multiplexed platforms.Competing Interest StatementCH and MR are employees of Promega Corporation, which provided reagents used in this study to the W{\"u}hr laboratory.National Institutes of Health (NIH), R35GM128813, T32GM007388Ludwig Cancer ResearchPrinceton Summer Undergraduate Research Program in Molecular and Quantitative \&amp; Computational BiologyPrinceton Summer Undergraduate Research Fellows in ChemistryPrinceton Catalysis Initiative</p>},
  year = {2025},
  journal = {bioRxiv},
  month = {07/2025},
  publisher = {Cold Spring Harbor Laboratory},
  url = {https://www.biorxiv.org/content/early/2025/07/18/2025.07.15.664461},
  doi = {10.1101/2025.07.15.664461},
}