基于环氧功能基团的杂化硅胶固定化酶反应器

A Novel Organic-Inorganic Hybrid Monolith for Trypsin Immobilization

在蛋白质组学研究中,为解决自由溶液酶解时间长、蛋白酶自降解等问题,各种类型的固定化酶反应器引起了人们的关注.采用溶胶凝胶法,在100μm内径毛细管内,以3-(甲基丙烯酰氧)丙基三甲氧基硅烷为功能单体,以四乙氧基硅烷为交联剂,制备了一种新型的杂化硅胶整体材料.并通过在整体柱表面由环氧功能团水解得到的二醇与胰蛋白酶(trypsin)的氨基进行一步反应,实现了胰蛋白酶固定化.利用该IMER,在47s内实现了牛血清白蛋白的酶解;经反相色谱分离和质谱鉴定,序列覆盖率在35%以上,与自由溶液酶解12h的效果相当.结果表明,基于亲水性杂化硅胶整体材料的IMER有望在蛋白质组学研究中发挥重要作用.

In proteomics, various immobilization enzyme reactors （IMER） have been put more attention for the realization of high throughput digestion. In this report, a novel organic-inorganic hybrid monolith based IMER was prepared in 100 ~tm i.d. capillary with 3-GLycidoxypropyLtrimethoxysiLane （GLYMO） as monomer and TetraethoxysiLane （TEOS） as crosslinker. Trypsin immobilization was achieved via the reaction between vicinal diol groups obtained from epoxy groups＇ hydrolysis and amino groups of trypsin. By this IMER, BSA was digested well in 47 s. After ~tRPLC-MS/MS analysis and database searching, beyond 35% sequence coverage was obtained, and the result was comparable to that of 12 h in solution digestion. The present IMER has shown an applicable potential for the high throughput digestion.