Fan Chao, Song Zifeng, Qin Weijie, Cai Yun, Qian Xiaohong
Graduate Department of Anhui Medical University, Hefei 230032, China.
Se Pu. 2013 May;31(5):423-8. doi: 10.3724/sp.j.1123.2013.01007.
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 protein bovine serum albumin (BSA), 1 min digestion led to the identification of 93 peptides, which covered 91% amino acid sequence of the protein. This immobilized trypsin 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 h solution digestion.
以原子转移自由基聚合（ATRP）合成的亲水性聚合物修饰硅胶微粒（HPMSM）为底物材料，制备了一种新型固定化胰蛋白酶。ATRP修饰使亲水性聚合物链紧密接枝在微粒表面，从而使胰蛋白酶负载量大幅增加，并使蛋白质和肽的非特异性吸附降至最低。因此，实现了超快速高效的蛋白质消化，且样品损失最小。对于标准蛋白牛血清白蛋白（BSA），1分钟的消化可鉴定出93个肽段，覆盖了该蛋白91%的氨基酸序列。这种固定化胰蛋白酶进一步成功应用于酵母复杂蛋白质样品的消化，3分钟消化后鉴定出666种蛋白质，超过了溶液消化12小时后鉴定出的蛋白质数量。
