This study presents a comparative analysis of three LysC endopeptidase homologues from Achromobacter lyticus (A. lyticus),Pseudomonas aeruginosa and Lysobacter enzymogenes for mass spectrometry-based proteomics. Utilizing a protein aggregation capture workflow with HeLa cell lysates, we assessed the enzymes’ cleavage specificity, digestion efficiency, and performance across various experimental conditions. Results showed that while all three LysC homologues exhibited high cleavage specificity at lysine residues, A. lyticus LysC outperformed the two others with superior peptide identification, digestion efficiency, and protein coverage, especially at shorter digestion times. Our experiments using a combination ofA. lyticusLysC and trypsin demonstrated the importance of employing LysC for significantly minimizing missed cleavage rates in tryptic digests, especially with regard to lysine-containing peptides. This study underscores A. lyticus LysC’s potential as an optimal choice for enhancing mass spectrometry-based proteomics.

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