Ricin Sequencing Using Multi-Enzyme Digestion Coupled with LC/MS/MS

Achieving high protein sequence coverage remains challenging in mass spectrometry-based analysis. However, conventional single-enzyme digestion approaches, due to their limited cleavage sites and preferences, often result in coverage blind spots in hydrophobic regions and sequences lacking specific residues, thereby restricting both the depth of identification and the reliability of results. In this study, we systematically investigated ricin A and B chains using a digestion strategy combining Trypsin, Chymotrypsin, and Elastase with LC/MS/MS analysis. Under single-enzyme conditions, the sequence coverage was 74.30% for chain A and 73.80% for chain B, whereas the combined multi-enzyme strategy increased the coverage to 93.21% (247/265 residues) for chain A and 97.72% (257/263 residues) for chain B. The complementary cleavage specificities of the three proteases effectively compensated for the blind spots of trypsin digestion in hydrophobic and non-K/R regions, thereby enabling near-complete sequence characterization of ricin A and B chains. This approach provides a robust methodological framework for the reliable identification of highly toxic proteins and offers broad applicability for biosafety applications.