Intensive Protein Digestion Using Cross-Linked Trypsin Aggregates in Proteomics Analysis

In this study, cross-linked enzyme aggregates of tissue-culture-grade trypsin (CLEAs-T) were prepared and introduced to the proteomics analysis instead of the expensive proteomics-grade trypsin, which benefits from the protein purification during the preparation of the CLEAs. Bovine serum albumin, lysozyme, bovine hemoglobin, and -casein were used as model proteins, and three intensive strategies (high-enzyme-concentration, high-temperature, and ultrasound-assisted digestion) were applied to the rapid and efficient digestion of model proteins by CLEAs-T. The peptide fragments were then identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, which revealed the higher sequence coverage and sharply reduced digestion time of these strategies compared with the conventional in-solution 12h digestion method. Morphology studies demonstrated that the excellent performance of CLEAs-T in proteomics analysis came from the scaffoldlike supermolecular structure, which provided a high resistance to the autolysis and denaturation of trypsin under high-enzyme-concentration, high-temperature, and ultrasound-assisted digestion.