Disulfide bond reduction within antibody mass spectrometry workflows is typically carried out using chemical reducing agents to produce antibody subunits for middle-down and middle-up analysis. In this contribution we offer an online electrochemical reduction method for the reduction of antibodies coupled with liquid chromatography (LC) and mass spectrometry (MS), reducing the disulfide bonds present in the antibody without the need for chemical reducing agents. An electrochemical cell placed before the analytical column and mass spectrometer facilitated complete reduction of NISTmAb inter- and intrachain disulfide bonds. Reduction and analysis were carried out under optimal solvent conditions using a trapping column and switching valve to facilitate solvent exchange during analysis. The level of reduction was shown to be affected by electrochemical potential, temperature and solvent organic content, but with optimization, complete disulfide bond cleavage was achieved. The use of an inline electrochemical cell offers a simple, rapid, workflow solution for liquid chromatography mass spectrometry analysis of antibody subunits.

中文翻译：

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NISTmAb 的在线电化学还原用于中间亚单位液相色谱-质谱分析

抗体质谱工作流程中的二硫键还原通常使用化学还原剂进行，以产生用于中下和中上分析的抗体亚基。在这篇文章中，我们提供了一种在线电化学还原方法，用于与液相色谱 (LC) 和质谱 (MS) 结合的抗体还原，无需化学还原剂即可还原抗体中存在的二硫键。放置在分析柱和质谱仪之前的电化学池有助于完全还原 NISTmAb 链间和链内二硫键。使用捕集柱和切换阀在最佳溶剂条件下进行还原和分析，以促进分析过程中的溶剂交换。显示还原水平受电化学电位、温度和溶剂有机物含量的影响，但通过优化，实现了完全的二硫键断裂。在线电化学池的使用为抗体亚基的液相色谱质谱分析提供了一种简单、快速的工作流程解决方案。