Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion.
Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion.
复制标题
DOI：
10.1002/pmic.200800591
复制
发表时间：
2009-04
期刊：
影响因子：
3.4
通讯作者：
Kim, Jungbae
中科院分区：
文献类型：
--
作者：
Kim, Byoung Chan;Lopez-Ferrer, Daniel;Lee, Sang-Mok;Ahn, Hye-Kyung;Nair, Sujith;Kim, Seong H.;Kim, Beom Soo;Petritis, Konstantinos;Camp, David G.;Grate, Jay W.;Smith, Richard D.;Koo, Yoon-Mo;Gu, Man Bock;Kim, Jungbae
一个稳定的和强大的胰蛋白酶为基础的生物催化系统的开发和展示的蛋白质组学应用。该系统利用包被有胰蛋白酶聚集体的聚合物纳米纤维用于固定化蛋白酶消化酶。在将胰蛋白酶的初始层共价连接到聚合物纳米纤维上后，高浓度的胰蛋白酶分子通过戊二醛处理与分层的胰蛋白酶交联。与用于共价胰蛋白酶固定化的常规方法相比，该方法产生了300倍的胰蛋白酶活性增加，并且证明是稳健的，因为在一年的重复回收后，它仍然保持高水平的活性。这种高度稳定形式的固定化胰蛋白酶是耐自溶，使重复digestion牛血清白蛋白超过40天，并成功地通过LC-MS/MS的肽鉴定。这种活性和稳定形式的固定化胰蛋白酶被成功地用于酵母蛋白质组提取物的消化，具有高重现性，并在较短的时间内比传统的蛋白质消化使用溶液相胰蛋白酶。最后，固定化的胰蛋白酶在暴露于其他酶（即胰凝乳蛋白酶）时对蛋白质水解具有抗性，这使得它适合用于“真实世界”的蛋白质组学应用。总的来说，具有胰蛋白酶聚集体涂层的生物催化纳米纤维被证明是蛋白质组学分析中重复和自动化蛋白质消化的有效方法。
A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of bovine serum albumin over 40 days and successful peptide identification by LC-MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e. chymotrypsin), which makes it suitable for use in “real-world” proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.
登录
查看更多内容
影响因子：
13.1
作者：
Lopez-Ferrer, D.;Canas, B.;Capelo, J. L.
通讯作者：
Capelo, J. L.
影响因子：
3.5
作者：
Kim, BC;Nair, S;Gu, MB
通讯作者：
Gu, MB
影响因子：
2.9
作者：
Krogh, TN;Berg, T;Hojrup, P
通讯作者：
Hojrup, P
影响因子：
7.4
作者：
YATES, JR;ENG, JK;SCHIELTZ, D
通讯作者：
SCHIELTZ, D
影响因子：
7.4
作者：
Shen, Yufeng;Hixson, Kim K.;Smith, Richard D.
通讯作者：
Smith, Richard D.