Enhanced Proteomics Analysis with a Novel Recombinant Chymotrypsin Analogue Engineered for High Cleavage Specificity

Chymotrypsin is widely used in shotgun proteomics owing to its orthogonal cleavage specificity relative to trypsin, which enhances sequence coverage of hydrophobic protein regions. However, commercial preparations often display variable cleavage specificity, trypsin contamination, and elevated missed cleavage rates, which can collectively reduce proteome coverage and data reproducibility. To address these limitations, we present a novel recombinant chymotrypsin (rChymoSelect) engineered for improved cleavage specificity and robustness in proteomics workflows. Benchmarking against standard bovine chymotrypsin revealed 97 % C terminal cleavages after tyrosine (Y), phenylalanine (F), and leucine (L) for rChymoSelect, compared with 72 % for the standard enzyme. This enhanced cleavage specificity reduced missed cleavages and increased peptide spectrum matches across charge states. Across 3,638 identified proteins, rChymoSelect yielded 22.2 % unique identifications compared with 8.2 % for standard chymotrypsin, while maintaining similar peptide length, m/z, and hydrophobicity distributions. Notably, rChymoSelect showed enriched recovery of mitochondrial proteins, consistent with its improved digestion of hydrophobic targets. The enzyme remained active in up to 6 M urea and achieved near maximal proteome coverage within 2 hours (only a 2.4 % gain after overnight digestion). Integration with data independent acquisition (DIA) increased total protein identifications from approximately 2,200 (DDA) to 3,200 (DIA), a 45 % gain, with rChymoSelect outperforming standard chymotrypsin by 16.6 to 22.4 % in peptide-spectrum matches and 4.6 to 6.2 % in protein identifications. These results establish rChymoSelect as an advanced tool with improved cleavage specificity that reduces analytical complexity and enhances the reliability of proteomic analysis, while expanding chymotryptic digestion to hydrophobic and high denaturant proteomics applications.