Robust immobilized enzyme reactor based on trimethylolpropane trimethacrylate organic monolithic matrix through thiol-ene click reaction

A novel immobilized enzyme reactor (IMER) based on trimethylolpropane trimethacrylate (TRIM) organic monolith was prepared by thiol-ene click reaction. The IMERs were made by fabricating TRIM monolith core in advance. There were residual double bonds on the surface of TRIM monolith, in which click reaction can be performed with the free thiol groups of trypsin at room temperature to bond the trypsin on the monolithic column without additionally introducing an active functional group. The enzyme activity was characterized by kinetic parameters K-m and V-max and determined by off-line chromatography. The performance of the IMERs was evaluated by digesting standard protein BSA and compared with in-solution digestion method. By using IMER 5 min, the sequence coverage rate of BSA was 79.41%, which was similar to that in-solution digestion for 12 h (82.7%). The applicability of the IMERs in complex samples was assessed by digesting crude protein extracts from egg white and mouse liver, identifying 28 (104 peptides) and 843 (3916 peptides) proteins, respectively. All results indicated that the IMERs have great potential in high-throughput proteomics analysis.