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色谱  0, Vol. Issue (): 0-0    DOI: 10.3724/SP.J.1123.2012.10030
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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
周廉淇1,张姣1,田芳1,张养军2,钱小红1
1. 北京蛋白质组研究中心
2. 国家生物医学分析中心仪器测试中心
Preparation of a Trypsin Immobilizd Reactor on Silver Wire Modified by Atom Transfer Radical Polymer and its Application in Proteome Identification
Lian-Qi ZHOU 2,
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摘要 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,我们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,并以其做为载体制备了一种新型的固定化酶反应器。采用标准蛋白质和质谱考察了银丝固定化酶反应器的酶解效率、重复性和回收率。结果表明:绒毛状聚合物修饰的银丝固定化酶反应器的酶解效率较高,酶解标准蛋白BSA 20 min后,肽段的氨基酸序列覆盖率可达93%,好于传统溶液酶切方法酶切16 h得到的79%的覆盖率;使用该固定化酶反应器在一个月内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%,显示出其具有良好的稳定性。另外MYO的回收率实验结果表明该固定化酶的回收率为87.67%。最后,银丝固定化酶反应器酶解腾冲嗜热菌全蛋白20 min所鉴定到的序列覆盖率和鉴定的蛋白数量与同样条件下溶液酶解16小时的结果接近,而零漏切位点肽段的比例更高。加之银丝固定化酶反应器容易分离的优点,表明银丝固定化酶反应器在蛋白质组学的应用中具有良好的前景。
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周廉淇
张姣
田芳
张养军
钱小红
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Abstract: Abstract: 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 using standard proteins and subsequent mass spectrometry (MS) analysis. Highly efficient digestion was achieved by using SW-Trypsin within only 20 minutes. Standard proteins 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. BSA was digested 8 times within a month by using the SW-Trypsin. The result of sequence coverage was between 89% to 97%, with an average sequence coverage of 94%, which showed that SW-Trypsin has good stability. In addition, The recovery test by using 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, respectively. The results of sequence coverage 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 is higher than that by in-solution digestion. Also, the SW-Trypsin is easily removed from digestion solution. Good performances of SW-Trypsin imply that it has a good prospect in the application in future proteomics research.
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收稿日期: 2012-11-05; 出版日期: 2012-11-23
通讯作者: 周廉淇   
引用本文:   
周廉淇,张姣,田芳等. 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[J]. 色谱, 0, (): 0-0.
ZHOU Lian-Qi,ZHANG Jiao,TIAN Fang et al. Preparation of a Trypsin Immobilizd Reactor on Silver Wire Modified by Atom Transfer Radical Polymer and its Application in Proteome Identification[J]. Chinese Journal of Chromatography, 0, (): 0-0.
 
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