原子转移自由基聚合修饰银丝固定化酶反应器的制备及在蛋白质组鉴定中的应用  

Preparation of a trypsin immobilized reactor on silver wire modified by atom transfer radical polymer and its application in proteome identification

作　　者：周廉淇[1,2] 张姣[2] 田芳[2] 张养军[2] 钱小红[2] 

机构地区：[1]安徽医科大学,安徽合肥230032 [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

出　　处：《色谱》2013年第4期355-361,共7页Chinese Journal of Chromatography

基　　金：国家重大科学计划项目(2012CB910603;2010CB912704);国家重大科学仪器设备开发专项项目(2011YQ030139;2011YQ06008408;2012YQ12004407);国家高技术研究发展计划项目(2012AA020202);国家自然科学基金项目(20735005;21275159)

摘　　要：针对传统溶液酶解存在的酶解时间较长、酶自切物干扰以及蛋白酶不能重复使用等缺陷,通过电子转移生成催化剂的原子转移自由基聚合法修饰银丝,并以其为载体制备了一种新型的固定化酶反应器。用质谱考察了银丝固定化酶反应器(SW-Trypsin)的酶解效率、重复性和回收率。结果表明:绒毛状聚合物修饰的SW-Trypsin的酶解效率较高,酶解标准蛋白牛血清白蛋白(BSA)20 min后,肽段的氨基酸序列覆盖率可达93%,高于传统溶液酶解方法酶解16 h所得79%的覆盖率。使用该固定化酶反应器于一个月内8次酶解BSA所得的氨基酸序列覆盖率在89%到97%之间,平均覆盖率为94%,显示出良好的稳定性。另外,该固定化酶反应器酶解马心肌红蛋白(MYO)的回收率为87.67%。最后,用SW-Trypsin酶解腾冲嗜热菌全蛋白20 min,所鉴定到的氨基酸序列覆盖率和蛋白数量与同样条件下溶液酶解16 h的结果接近,且零漏切位点肽段的比例更高。加之容易分离的优点,SW-Trypsin在蛋白质组学的应用中具有良好的前景。The routine proteolysis of proteins is performed in solution,but it suffers from drawbacks such as long incubation time,enzyme autodigestion,and non-reusability.Therefore we here demonstrated that trypsin could be immobilized on silver wire modified by atom transfer radical polymerization to prepare a new kind of enzyme immobilized reactor.The digestion efficiency,repeatability and recovery of the silver wire-trypsin reactor（SW-Trypsin） were evaluated by mass spectrometry（MS） analysis.Highly efficient digestion was achieved by using SW-Trypsin within only 20 min.Standard protein could be almost completely digested with sequence coverage up to 93%,which is higher than that of 79% sequence coverage obtained by in-solution digestion for 16 h.Bovine serum albumin（BSA） was digested eight times within a month by using the SW-Trypsin.The results of sequence coverage were between 89% to 97%,with an average sequence coverage of 94%,which showed that SW-Trypsin had good stability.In addition,the recovery test by using myoglobin（MYO） showed that the recovery rate was 87.67%.At last,the extract from Tengchong thermophilic bacteria was digested by SW-Trypsin in 20 min and in-solution trypsin in 16 h.The results of sequence coverages and the number of identified proteins were similar.Moreover,the ratio of the number of peptides with zero missed cleavages to the number of all identified peptides by using SW-Trypsin was higher than that by in-solution digestion.Also,the SW-Trypsin was easily removed from the digestion solution.Good performances of SW-Trypsin implied that it has a good prospect in the application in future proteomics research.

关 键 词：原子转移自由基聚合反应 蛋白质组学 质谱 固定化酶反应器 银丝 

分 类 号：O658[理学—分析化学]