溶胶凝胶膜包埋的胰蛋白酶用于高通量蛋白质肽谱分析  

Trypsin Immobilized in Sol-gel for High-throughput Protein Peptide Mapping

作　　者：栾明明[1] 邬建敏[1] 

机构地区：[1]浙江大学化学系,杭州310027

出　　处：《分析化学》2006年第12期1707-1710,共4页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金资助项目(No.20277031)

摘　　要：采用溶胶凝胶包埋法在96孔板中固定胰蛋白酶,制备高通量的酶解反应器。考察了四乙氧基硅烷(TEOS)与甲基三甲氧基硅烷(MTMS)的不同比例对凝胶膜的成胶速度、机械强度、透明度、固定酶的活性及稳定性的影响。以牛血清白蛋白(BSA)为目标蛋白,比较了溶胶凝胶固定化酶与溶液态酶的稳定性和催化活性,用HPLC对BSA的酶解产物进行了肽谱分析,比较了二者的酶解效率。结果表明,TEOS与MTMS比例为1∶1时,凝胶膜有很好的成胶速度、机械强度、透明度;包埋态酶有很高的活性和稳定性。HPLC对肽段的分析结果表明,固定化酶的酶解效率优于溶液态酶。A high-throughput tryptic reactor was prepared by coating a trypsin-containing gel on a 96-well microtiter plate. The trypsin-encapsulated gel was prepared by the sol-gel method, incorporating methyltrimethoxysilane （MTMS） into tetraethoxysilane （TEOS） at various ratios. The effect of MTMS ratio on gelation speed, meehanical strength, transparency, the activity and stability of enzyme immobilized was studied. Furthermore, the digestion efficiency for bovine serum albumin （BSA） by immobilized enzyme and free enzyme was also compared. The peptide fragments maps were acquired by high performance liquid chromatography. Result showed that trypsin embedded in sol-gel membrane formed by MTMS and TEOS （ 1 ： 1 ） gave the best properties of gel, the highest enzymatic activity and higher digestion efficiency.

关 键 词：溶胶凝胶 固定化胰蛋白酶 蛋白质酶解 肽谱分析 

分 类 号：Q51-33[生物学—生物化学]