Disulfide bonds are critical linkages for maintaining protein structure and enzyme activity. These linkages, however, can limit peptide sequencing efforts by mass spectrometry (MS) and often require chemical reduction and alkylation. Under such conditions, information regarding cysteine connectivity is lost. Online partial disulfide reduction within the electrospray (ESI) source has recently been established as a means to identify complex cysteine linkage patterns in a liquid chromatography-MS experiment without the need for sample pre-treatment. Corona discharge (CD) is invoked as the causative factor of this in-source reduction (ISR); however, evidence remains largely circumstantial. In this study, we demonstrate that instrumental factors—nebulizing gas, ESI capillary material, organic solvent content, ESI spray needle-to-MS distance—all modulate the degree of reduction observed for the single disulfide in oxytocin, further implicating CD in ISR. Rigorous analysis of solution conditions, however, reveals that corona discharge alone can induce only minor disulfide reduction. We establish that CD-ESI of peptide solutions containing formic acid or its conjugate base results in a dramatic increase in disulfide reduction. It is also determined that ISR is exacerbated at low pH for complex peptides containing multiple disulfide bonds and possessing higher-order structure, as well as for a small protein. Overall, our results demonstrate that ESI of formate/formic acid–containing solutions under corona discharge conditions facilitates disulfide ISR, likely by a similar reduction pathway measured in γ-radiolysis studies nearly three decades ago.

中文翻译：

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含甲酸溶液的电晕放电电喷雾电离可实现二硫键的源内还原

二硫键是维持蛋白质结构和酶活性的关键键。但是，这些连接可能会限制通过质谱（MS）进行的肽测序工作，并且经常需要化学还原和烷基化。在这种情况下，有关半胱氨酸连接性的信息会丢失。最近，已经建立了电喷雾（ESI）源中在线部分二硫化物还原的方法，该方法可用于鉴定液相色谱-MS实验中复杂的半胱氨酸键合模式，而无需进行样品预处理。电晕放电（CD）被称为造成这种源内减少（ISR）的原因。但是，证据仍然基本上是间接的。在这项研究中，我们证明了仪器因素-雾化气体，ESI毛细管材料，有机溶剂含量，ESI喷雾针距MS的距离-都可调节催产素中单个二硫键的还原度，从而进一步将CD牵连到ISR中。但是，对溶液条件的严格分析表明，单独的电晕放电只能引起少量的二硫化物还原。我们建立了包含甲酸或其共轭碱的肽溶液的CD-ESI导致二硫化物还原的急剧增加。还确定了对于包含多个二硫键并具有高阶结构的复杂肽以及小的蛋白质，在低pH下ISR会加剧。总的来说，我们的结果表明，在电晕放电条件下，含甲酸/甲酸的溶液的ESI促进了二硫化物ISR的产生，这很可能是通过近三十年前在γ射线辐照研究中测得的类似还原途径进行的。