新型亲水聚合物修饰硅胶颗粒固定化酶的制备及在蛋白质组鉴定中的应用  

A novel immobilized trypsin on hydrophilic polymer modified silica beads for proteome characterization

作　　者：范超[1,2] 宋子凤[2] 秦伟捷[2] 蔡耘[2] 钱小红[2] 

机构地区：[1]安徽医科大学研究生学院,安徽合肥230032 [2]军事医学科学院放射与辐射医学研究所,北京蛋白质组研究中心,蛋白质组学国家重点实验室,北京102206

出　　处：《色谱》2013年第5期423-428,共6页Chinese Journal of Chromatography

基　　金：国家重大科学计划项目(2013CB911204);国家重大科学仪器设备开发专项项目(2011YQ09000504);国家自然科学基金项目(21275005,21235001)

摘　　要：采用原子转移自由基聚合法制备了亲水聚合物修饰的硅胶颗粒作为一种新型固定化酶载体,在实现胰蛋白酶高密度固定的同时,显著降低了载体材料非特异性吸附导致的样品损失。因此,该固定化酶材料兼具高酶解效率和高回收率的特性。以标准蛋白质牛血清白蛋白(BSA)为样本,使用该固定化酶1 min即可完成酶解,鉴定到肽段对BSA的氨基酸序列覆盖率可达90%以上。该固定化酶材料成功应用于酵母菌全蛋白质复杂样本的酶解,从3min酶解产物中鉴定到666个蛋白质,超过同样条件下溶液酶解12 h的鉴定结果。Abstract： A new type of immobilized trypsin was prepared using hydrophilic polymer modified silica microparticles （HPMSM） synthesized by atom transfer radical polymerization （ATRP） as the substrate material. ATRP modification led to densely packed hydrophilic polymer chains grafted on the microparticles surface which resulted in largely increased trypsin loading amount and minimized the nonspecific adsorption of proteins and peptides. Therefore, ultra-fast and highly efficient protein digestion was achieved with minimized sample loss. For standard pro- tein bovine serum albumin （ BSA）, 1 rain digestion led to the identification of 93 peptides, which covered 91% amino acid sequence of the protein. This immobilized tt^jpsin was further successfully applied to the digestion of complex protein samples from yeast and 666 proteins were identified after 3 min digestion, which exceeded the number of identified proteins after 12 1/ solution digestion.

关 键 词：固定化酶 蛋白质组学 原子转移自由基聚合反应 

分 类 号：O658[理学—分析化学]