Self-assembly synthes is of trypsin-immobilized monolithic microreactor for fast and efficient proteolysis

Fast and highly efficient digestion of proteins is essential for high-throughput proteomic analysis. Herein, a facile approach was developed for self-assembly preparation of trypsin-immobilized capillary monolithic column and its application as an immobilized enzyme microreactor (IMER) for fast and highly efficient proteolysis was described. The performance of the trypsin-immobilized monolithic enzyme microreactor was evaluated by in-situ digestion of model proteins. The results showed that the trypsin-immobilized monolithic enzyme microreactor had much higher tryptic digestion efficiency than the free trypsin in solution, where the coverage of peptide sequences by mass spectrometry (MS)-based analysis could bear comparison with the free one, while the digestion time was dramatically shortened from 12 h to 16 s. Furthermore, the trypsin-immobilized monolithic enzyme microreactor also exhibited good practicability to complex human serum sample, in which the total of 45 peptides from human serum albumin (HSA) matched with sequence coverage of 75% were precisely identified. The successful application demonstrated the promising potential of the trypsin-immobilized capillary monolithic column as the IMER in high-throughput proteomic analysis.