Department of Biochemistry and Molecular Biology, University of Southern Denmark , Campusvej 55, Odense M DK-5230, Denmark.
Anal Chem. 2014 Jan 7;86(1):340-5. doi: 10.1021/ac403269a. Epub 2013 Dec 23.
Characterization of disulfide bond-containing proteins by hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) requires reduction of the disulfide bonds under acidic and cold conditions, where the amide hydrogen exchange reaction is quenched (pH 2.5, 0 °C). The reduction typically requires a high concentration (>200 mM) of the chemical reducing agent Tris(2-carboxyethyl)phosphine (TCEP) as its reduction rate constant is decreased at low pH and temperature. Serious adverse effects on chromatographic and mass spectrometric performances have been reported when using high concentrations of TCEP. In the present study, we explore the feasibility of using electrochemical reduction as a substitute for TCEP in HDX-MS analyses. Our results demonstrate that efficient disulfide bond reduction is readily achieved by implementing an electrochemical cell into the HDX-MS workflow. We also identify some challenges in using electrochemical reduction in HDX-MS analyses and provide possible conditions to attenuate these limitations. For example, high salt concentrations hamper disulfide bond reduction, necessitating additional dilution of the sample with aqueous acidic solution at quench conditions.
通过质谱监测的氢/氘交换法（HDX-MS）对含二硫键的蛋白质进行特性分析，需要在酸性和低温条件下还原二硫键，此时酰胺氢交换反应被猝灭（pH 2.5，0°C）。还原通常需要高浓度（>200mM）的化学还原剂三（2-羧乙基）膦（TCEP），因为其还原速率常数在低 pH 和温度下会降低。当使用高浓度的 TCEP 时，已经报道了对色谱和质谱性能的严重不利影响。在本研究中，我们探讨了电化学还原作为 HDX-MS 分析中 TCEP 的替代方法的可行性。我们的结果表明，通过在 HDX-MS 工作流程中实施电化学池，可以轻松实现有效的二硫键还原。我们还确定了在 HDX-MS 分析中使用电化学还原时存在的一些挑战，并提供了可能的条件来减轻这些限制。例如，高盐浓度会阻碍二硫键还原，因此需要在猝灭条件下用酸性水溶液对样品进行额外稀释。
