Preparation and evaluation of an enzymatic microreactor based on HILIC matrix for digestion and identification of proteins

In the present study, a new enzymatic microreactor is developed by immobilizing trypsin on the matrix-HILIC (hydrophilic interaction chromatography). The influences of different types of buffer solutions (borate, Tris·HCl, and phosphate), their concentrations and pH values on the immobilizing rate and the activity of immobilized trypsin were investigated. The optimal condition for off-line immobilization of trypsin on matix-HILIC was Tris·HCl buffer (20 mmol/L, pH 8. 0), and then, under the optimal condition, an enzymatic microreactor was prepared by trypsin dynamically immobilized on HILIC matrix which was packed in a Peeksil column. It can be found that the microreactor possessed high hydrolytic activity according to the hydrolysis results of N-α-benzoyl-DL-arginine P-nitroanilide (BAPNA), a substrate of trypsin. Moreover, bovine serum albumin (BSA), cytochrome C and myoglobin as standard proteins were chosen to characterize the enzymolysis of the microreactor demonstrating the high enzymolysis efficiency by this enzymatic microreactor. Besides, the deactivated trypsin can be easily eluted from the matrix-HILIC to re-immobilize trysin, which is highly cost-effective in the experiment. All the results show that the new enzymatic microreactor associated with HPLC-MS techniques can realize online enzymolysis of proteins, and the time to analyze and identify the proteins is greatly shortened.