色谱

色谱 ›› 2012, Vol. 30 ›› Issue (06): 549-554.DOI: 10.3724/SP.J.1123.2012.02006

• 研究论文 • 上一篇    下一篇

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

宋子凤1,2, 张庆林2, 张养军2*, 秦伟捷2*, 钱小红2   

  1. 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, 北京 102206
  • 收稿日期:2012-02-06 修回日期:2012-03-07 出版日期:2012-06-28 发布日期:2012-06-20
  • 通讯作者: 张养军,博士,硕士生导师,研究方向为蛋白质组学的新方法新技术.秦伟捷,博士,副研究员,研究方向为蛋白质组学的新方法新技术.
  • 基金资助:

    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, 2011YQ06008408, 2011YQ09000504)和国家自然科学基金项目(Nos. 20735005, 20905077).

Trypsin immobilization on silica beads modified by squamous polymer for ultra fast and highly efficient proteome digestion

SONG Zifeng1,2, ZHANG Qinglin2, ZHANG Yangjun2*, QIN Weijie2*, QIAN Xiaohong2   

  1. 1. Guangxi Medical University, Nanning 530021, China; 2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China
  • Received:2012-02-06 Revised:2012-03-07 Online:2012-06-28 Published:2012-06-20

PDF

415

被引次数

26

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间。使用标准蛋白质对固定化酶的酶解效率进行了考察,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1 min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2 min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12 h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。

关键词: 蛋白质组学, 鳞片状聚合物修饰硅胶, 胰蛋白酶固定, 原子转移自由基聚合反应, 制备

Abstract: 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 prepared by surface initiated atom transfer radical polymerization (SI-ATRP), which leaded to surface 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 efficient digestion was achieved in only 1-2 min digestion. Furthermore, the SPMSB-Trypsin exhibited both good stability and excellent recovery, therefore can be applied in proteomic research in the future.

Key words: preparation, proteomics, squamous polymer modified silica bead (SPMSB), trypsin immobilization, atom transfer radical polymerization (ATRP)