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新型鳞片状聚合物修饰硅胶填料固定化酶的制备及其在蛋白质组鉴定中的应用

ISSN号：1000-8713
期刊名称：色谱
时间：0
页码：549-554
分类：TQ316.6[化学工程—高聚物工业]
作者机构：[1]广西医科大学,广西南宁530021, [2]蛋白质组学国家重点实验室北京蛋白质组研究中心军事医学科学院放射与辐射医学研究所,102206
相关基金：国家重大科学计划项目（Nos.2012CB910603,2010CB912704）、国家重大科学仪器设备开发专项项目（Nos.2011YQ030139,201IYQ06008408.2011YQ09000504）和国家自然科学基金项151（Nos.20735005.20905077）.
相关项目：金纳米材料和生物质谱用于糖基化蛋白质/糖肽检测、富集及鉴定的新方法研究

关键词：原子转移自由基聚合反应, 鳞片状聚合物修饰硅胶, 制备, 胰蛋白酶固定, 蛋白质组学, atom transfer radical polymerization （ATRP） , squamous polymer modified silicabead （SPMSB） , preparation： trypsin immobilization~ proteomics

中文摘要：

利用原子转移自由基聚合法制备了鳞片状聚合物修饰的硅胶填料，将其作为一种新型的固定化酶载体，实现了蛋白酶的高密度固定，从而明显缩短了复杂蛋白质样品的酶解时问。使用标准蛋白质对固定化酶的酶解效率进行r考察，结果表明：鳞片状聚合物修饰的新型固定化酶硅胶填料具有较高的酶解效率，酶解标准蛋白质1min后，鉴定到肽段的氨基酸序列覆盖率可达95％以上。将该固定化酶硅胶填料成功应用于大肠杆菌全蛋白质的酶解，从2min酶解肽段的混合物中鉴定到的蛋白质数量超过同样条件下溶液酶解12h的结果。另外，该固定化酶硅胶填料可以重复使用，其酶解效率具有良好的稳定性和重现性；该固定化酶具有较好的样品回收率，因而可以应用于蛋白质组学研究中。

英文摘要：

Currently, the shotgun based strategy has been widely applied in proteomic research. In this strategy, protein identification relied on the identification of the corresponding proteolytic peptides. Therefore, rapid and efficient protein digestion is crucial for accurate protein identification and characterization. Even though traditional free protein digestion in solution has been widely adopted, it had a few inherent disadvantages including long incubation time, incomplete digestion and non-reusability of the protease. In this work, we developed a new type of trypsin immobilized on squamous polymer modified silica bead （SPMSB） for ultra fast and highly efficient protein digestion. The squamous polymer coated silica beads were pre- pared by surface initiated atom transfer radical polymerization （ SI-ATRP）, which leaded to sur- face confined growth of non-crosslinked polymer chains on the surface of the silica beads for trypsin immobilization. The digestion efficiency of the obtained SPMSB-trypsin was evaluated using both standard proteins and complex protein extracts obtained from E. coli. Highly effi- cient digestion was achieved in only l -2 min digestion. Furthermore, the SPMSB-Trypsin exhibited both good stability and excellent recovery, therefore can be applied in proteomic research in the future.