Protein Digestion Using Immobilized Enzyme and a Microscale Vibration Unit for Structural Analysis of Phospholipase A2 by Mass Spectrometry

For mass spectroscopy (MS)-based protein structural analyses, effective digestion by proteases is one of the key steps. In this study, we developed a protein digestion method using immobilized enzyme and a microscale vibration unit. Phospholipase A2 was applied as a model substrate to evaluate the new procedure due to its rigid structure and resistance to protease digestion. Digested substrates were analyzed by MS, and cleavage at all expected recognition sites was evaluated. In relation to conventional liquid-phase digestion, the number of matched peptides and sequence coverage improved significantly from 6 to 9, and 41% to 69%, respectively. These results support the efficacy of our novel method in proteomics applications, including protein structural and post-translational modification analyses.