Electrochemistry-assisted top-down characterization of disulfide-containing proteins.
Electrochemistry-assisted top-down characterization of disulfide-containing proteins.
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DOI：
10.1021/ac300106y
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发表时间：
2012-04-17
影响因子：
7.4
通讯作者：
Chen, Hao
中科院分区：
文献类型：
--
作者：
Zhang, Yun;Cui, Weidong;Zhang, Hao;Dewald, Howard D.;Chen, Hao
蛋白质中的共价二硫键连接对于基于质谱 (MS) 的自上而下的蛋白质结构分析来说是一个重要的挑战，因为它降低了 MS/MS 解离的主链裂解效率。本研究提出了一种通过在线电化学 (EC) 与自上而下的 MS 方法相结合来解决这一关键问题的策略。在这种方法中，蛋白质在电化学电池中进行电解还原以破坏二硫键，然后在线电离成气态离子，以便通过电子捕获解离 (ECD) 和碰撞诱导解离 (CID) 进行分析。蛋白质的电化学还原可以消除二硫键的限制，并且还导致所得蛋白质离子的电荷数增加。结果，序列覆盖度显着增强，如 β-乳球蛋白 A（电解还原前后分别有 24 次主链裂解和 73 次主链裂解）和溶菌酶（电解还原前后分别有 5 次和 66 次主链裂解）为例。该方法快速且不需要化学还原剂，这将对高通量蛋白质组学研究产生重要影响。
Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then online ionized into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by β-lactoglobulin A (24 vs. 73 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs. 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research.
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DOI：
10.1039/c1an15286f
发表时间：
2011-10-07
期刊：
The Analyst
影响因子：
--
作者：
Cui W;Rohrs HW;Gross ML
通讯作者：
Gross ML
影响因子：
15
作者：
Kelleher, NL;Lin, HY;McLafferty, FW
通讯作者：
McLafferty, FW
影响因子：
64.8
作者：
Tran, John C.;Zamdborg, Leonid;Ahlf, Dorothy R.;Lee, Ji Eun;Catherman, Adam D.;Durbin, Kenneth R.;Tipton, Jeremiah D.;Vellaichamy, Adaikkalam;Kellie, John F.;Li, Mingxi;Wu, Cong;Sweet, Steve M. M.;Early, Bryan P.;Siuti, Nertila;LeDuc, Richard D.;Compton, Philip D.;Thomas, Paul M.;Kelleher, Neil L.
通讯作者：
Kelleher, Neil L.
影响因子：
16.6
作者：
Breuker, K;McLafferty, FW
通讯作者：
McLafferty, FW
影响因子：
7.4
作者：
Mikhailov, Victor A.;Iniesta, Jesus;Cooper, Helen J.
通讯作者：
Cooper, Helen J.