Characterization of Schizophyllum commune Metacaspase Autoproteolysis and Cleavage Sites Using LC-MS

Metacaspases are cysteine-dependent proteases found in fungi, plants, and protozoa that require calcium for activity. These enzymes cleave proteins specifically after arginine or lysine residues and play key roles in programmed cell death and innate immune responses. A major challenge in studying purified metacaspases is their tendency to undergo autoproteolysis, which complicates the analysis of their optimal activity and structural conformation upon calcium binding. Previous studies have shown that metacaspases from various species undergo autoproteolytic cleavage at conserved lysine and arginine sites. Liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for characterizing cleavage fragments, enabling the identification of primary cleavage sites based on molecular weight and fragmentation pattern. Proteins were chemically extracted from SDS-PAGE gels and further digested with chymotrypsin. The effectiveness of SDS-PAGE digestion for protein extraction was validated using a Bio-Rad assay. The samples were then analyzed by LC-MS to generate a chromatogram. Peaks of interest were selected, and their MS data were extracted and analyzed using Expasy's FindPept tool to identify specific peptide fragments. To verify the LC-MS method, a commercial, pre-digested BSA solution was analyzed. The MS data confirmed alignment with specific fragments from the BSA digest, validating the accuracy of the method.