Preparation And Study Of Novel Immobilized Enzyme Reactors For Proteomics

A novel immobilized enzyme reactor(IMER) based on trimethylolpropane trimethacrylate(TRIM) organic monolith was prepared by“thiol-ene”click reaction.The IMERs was made by fabricating TRIM monolith core in advance.Then trypsin was bonded to the monolithic column by“thiol-ene”click reaction with the free thiol groups on the surface of the TRIM monolith at room temperature without additionally introducing an active functional group.The enzyme activity was characterized by kinetic parameters K_m and V_maxand determined by off-line chromatography.The performance of the IMERs was evaluated by digesting standard protein BSA and compared with in-solution digestion methods.By using IMER 5 min,the sequence coverage rate of BSA was 79.41%,which was similar to that in-solution digestion for12 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(104peptides)and 843(3916 peptides)proteins,respectively.All results indicate that the IMERs have great potential in high-throughput proteomics analysis.Trypsin was successfully immobilized on the aminated mesoporous molecular sieve SBA-15 by glutaraldehyde coupling.SBA-15,as the fixed support of trypsin,has a strong adsorption capacity to the enzyme itself.In addition,the trypsin was fixed by glutaraldehyde coupling,which not only improved the immobilization amount of the enzyme,but also improved the activity and stability of the enzyme.The enzymatic hydrolysis of SBA-15 molecular sieve in solution has some disadvantages,such as difficult recovery of molecular sieve,partial loss and difficult separation of products.Therefore,the above problems can be solved by fixing the molecular sieve SBA-15.A new type of SBA-15-NH₂ doped organic-nanoparticle hybrid monolithic column immobilized enzyme reactor was prepared in this paper.The kinetic parameters K_m and V_max of trypsin were characterized by offline chromatography HPLC.IMERs prepared by this method were stable,and the enzyme activity remained stable after repeated use of IMERs for 30 times or storage in the refrigerator at 4? for 3 months.Through data analysis,64 unique peptides were identified and the sequence coverage was 73.8%.The online denaturation and digestion were integrated into a chip for the online denaturation and digestion of mouse liver protein extract.A total of 734 proteins and 3067 peptide segments were identified.The results show that IMERs have great application potential in high-throughput proteomics analysis.