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

查看全文 作　　者：WANG [1,2]ShanShan;LI [1]Qin;LU [1]XueFei;LIU [1]Tong;ZHANG [1]WeiBo;GENG [1]LiNa;DENG [1]YuLin 机构地区：[1]School of Life Science and Technology, Beijing Institute of Technology, Beijing 100081, China;[2]Analysis and Test Center, Shandong Academy of Sciences, Jinan 250014, China 出　　处：《Chinese Science Bulletin》索引2010年第55卷第10期,共5页 基　　金：supported by the Ministry of Science and Technology of China (Grant No.2006BAK03A07);National Natural Science Foundation of China (Grant No.20705005) 摘　　要：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. 关 键 词：酶催化反应 动态矩阵 蛋白质 评价鉴定 胰蛋白酶活性 酶固定化 消化 制备