Comparative Analysis of Lysine-Specific Peptidases for Optimizing Proteomics Workflows

This study presents a comparative analysis of three LysC endopeptidases homologues from Achromobacter lyticus, Pseudomonas aeruginosa, and Lysobacter enzymogenes for mass spectrometry-based proteomics. Utilizing a protein aggregation capture (PAC) workflow with HeLa cell lysate, 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 its superior peptide identification, digestion efficiency, and protein coverage, especially at shorter digestion times. Combination of A. lyticus LysC and Trypsin demonstrated the importance of employing LysC for significantly minimizing missed cleavage rates in tryptic digests. This study underscores A. lyticus LysC's potential as an optimal choice for enhancing mass spectrometry-based proteomics.