Trabjerg Esben, Jakobsen Rasmus U, Mysling Simon, Christensen Søren, Jørgensen Thomas J D, Rand Kasper D
Department of Pharmacy, University of Copenhagen , Universitetsparken 2, Copenhagen E, DK-2100, Denmark.
Department of Biologics, H. Lundbeck A/S , Ottiliavej 9, Valby, DK-2500, Denmark.
Anal Chem. 2015 Sep 1;87(17):8880-8. doi: 10.1021/acs.analchem.5b01996. Epub 2015 Aug 18.
Analysis of disulfide-bonded proteins by hydrogen/deuterium exchange mass spectrometry (HDX-MS) requires effective and rapid reduction of disulfide bonds before enzymatic digestion in order to increase sequence coverage. In a conventional HDX-MS workflow, disulfide bonds are reduced chemically by addition of a reducing agent to the quench solution (e.g., tris(2-carboxyethyl)phosphine (TCEP)). The chemical reduction, however, is severely limited under quenched conditions due to a narrow time window as well as low pH and temperature. Here, we demonstrate the real-world applicability of integrating electrochemical reduction into an online HDX-MS workflow. We have optimized the electrochemical reduction efficiency during HDX-MS analysis of two particularly challenging disulfide stabilized proteins: a therapeutic IgG1-antibody and nerve growth factor-β (NGF). Several different parameters (flow rate and applied square wave potential, as well as the type of labeling and quench buffer) were investigated, and the optimized workflow increased the sequence coverage of NGF from 46% with chemical reduction to 99%, when electrochemical reduction was applied. Additionally, the optimized workflow also enabled a similar high sequence coverage of 96% and 87% for the heavy and light chain of the IgG1-antibody, respectively. The presented results demonstrate the successful electrochemical reduction during HDX-MS analysis of both a small exceptional tightly disulfide-bonded protein (NGF) as well as the largest protein attempted to date (IgG1-antibody). We envision that online electrochemical reduction is poised to decrease the complexity of sample handling and increase the versatility of the HDX-MS technique.
通过氢/氘交换质谱法（HDX-MS）分析二硫键连接的蛋白质，需要在酶解之前有效且快速地还原二硫键，以提高序列覆盖率。在传统的HDX-MS工作流程中，通过向淬灭溶液中添加还原剂（例如三（2-羧乙基）膦（TCEP））来化学还原二硫键。然而，由于时间窗口狭窄以及低pH和低温，化学还原在淬灭条件下受到严重限制。在此，我们展示了将电化学还原整合到在线HDX-MS工作流程中的实际适用性。我们在对两种特别具有挑战性的二硫键稳定蛋白（一种治疗性IgG1抗体和神经生长因子-β（NGF））进行HDX-MS分析期间，优化了电化学还原效率。研究了几个不同的参数（流速和施加的方波电位，以及标记和淬灭缓冲液的类型），当应用电化学还原时，优化后的工作流程将NGF的序列覆盖率从化学还原时的46%提高到了99%。此外，优化后的工作流程还分别使IgG1抗体的重链和轻链的序列覆盖率达到了相似的高水平，分别为96%和87%。所呈现的结果表明在HDX-MS分析过程中，对于一个小的、异常紧密二硫键连接的蛋白（NGF）以及迄今为止尝试分析的最大蛋白（IgG1抗体），电化学还原均取得了成功。我们设想在线电化学还原有望降低样品处理的复杂性，并提高HDX-MS技术的通用性。
