Song Zifeng, Zhang Qinglin, Zhang Yangjun, Qin Weijie, Qian Xiaohong
Guangxi Medical University, Nanning 530021, China.
Se Pu. 2012 Jun;30(6):549-54. doi: 10.3724/sp.j.1123.2012.02006.
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 sufface 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. coil. Highly efficient digestion was achieved in only 1 - 2 mm digestion. Furthermore, the SPMSB-Trypsin exhibited both good stability and excellent recovery, therefore can be applied in proteomic research in the future.
目前，基于鸟枪法的策略已广泛应用于蛋白质组学研究。在该策略中，蛋白质鉴定依赖于相应蛋白水解肽段的鉴定。因此，快速高效的蛋白质消化对于准确的蛋白质鉴定和表征至关重要。尽管传统的溶液中自由蛋白质消化方法已被广泛采用，但它存在一些固有缺点，包括孵育时间长、消化不完全以及蛋白酶不可重复使用。在这项工作中，我们开发了一种新型的固定在鳞状聚合物修饰硅胶珠（SPMSB）上的胰蛋白酶，用于超快速和高效的蛋白质消化。通过表面引发原子转移自由基聚合（SI-ATRP）制备了鳞状聚合物包覆的硅胶珠，这导致在硅胶珠表面形成非交联聚合物链的表面受限生长，用于固定胰蛋白酶。使用标准蛋白质和从大肠杆菌中获得的复杂蛋白质提取物评估了所得SPMSB-胰蛋白酶的消化效率。仅在1 - 2分钟的消化时间内就实现了高效消化。此外，SPMSB-胰蛋白酶表现出良好的稳定性和出色的回收率，因此可在未来的蛋白质组学研究中应用。
