The ratio between reduced and oxidized thiols, mainly glutathione and oxidized glutathione, is one of the biomarkers for the evaluation of oxidative stress. The accurate measurement of thiol concentrations is challenging because reduced thiols are easily oxidized during sample manipulation. Derivatization is commonly used to protect thiols from oxidation. The objective of this work was to systematically compare two cell-permeable derivatizing agents: N-ethyl maleimide (NEM) and (R)-(+)-N-(1-phenylethyl)maleimide (NPEM) in terms of derivatization efficiency, ionization enhancement, side product formation, reaction selectivity for thiols, pH dependence of the reaction, and derivative stability. All thiol measurements and the characterization of side products were performed using a biphenyl reversed phase liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Four thiols, cysteine (CYS), homocysteine, N-acetylcysteine (NAC), and glutathione (GSH), were used for the evaluation. Using 1:10 ratio of thiol:derivatizing agent, complete derivatization was obtained within 30 min for both agents tested with the exception of CYS-NEM, where 97% efficiency was obtained. The more hydrophobic NPEM provided better ionization of the thiols, with enhancement ranging from 2.1x for GSH to 5.7x for CYS in comparison to NEM. NPEM derivatization led to more extensive side reactions, such as double derivatization and ring opening, which hindered the accurate measurement of the thiol concentrations. Both NEM and NPEM also showed poor stability of CYS derivative due to its time-dependent conversion to cyclic cysteine-maleimide derivative. Both reagents also showed significant reactivity with amine-containing metabolites depending on the pH used during derivatization, but overall NEM was found to be more selective towards thiol group than NPEM. Taking into account all evaluation criteria, NEM was selected as a more suitable reagent for the thiol protection and derivatization, but strict control of pH 7.0 is recommended to minimize the side reactions. This work illustrates the importance of the characterization of side products and derivative stability during the evaluation of thiol derivatizing agents and contributes fundamental understanding to improve the accuracy of thiol determinations. The key sources of errors during maleimide derivatization include the derivatization of amine-containing metabolites, poor derivative stability of certain thiols (CYS and NAC), and the side reactions especially if ring opening of the reagent is not minimized. Graphical abstract.

中文翻译：

---

使用液相色谱-质谱法比较N-乙基马来酰亚胺和N-（1-苯乙基）马来酰亚胺用于生物硫醇的衍生化

还原的和氧化的硫醇（主要是谷胱甘肽和氧化的谷胱甘肽）之间的比率是用于评估氧化应激的生物标记之一。巯基浓度的准确测量具有挑战性，因为在样品处理过程中还原的巯基很容易被氧化。衍生化通常用于保护硫醇免受氧化。这项工作的目的是系统化地比较两种可透过细胞的衍生剂：N-乙基马来酰亚胺（NEM）和（R）-（+）-N-（1-苯基乙基）马来酰亚胺（NPEM）的衍生效率，离子化程度增强，副产物形成，对硫醇的反应选择性，反应的pH依赖性和衍生物稳定性。使用联苯反相液相色谱-高分辨率质谱法（LC-HRMS）进行所有硫醇测量和副产物表征。评估使用了四个硫醇，半胱氨酸（CYS），高半胱氨酸，N-乙酰半胱氨酸（NAC）和谷胱甘肽（GSH）。使用1:10的硫醇：衍生化剂比例，在30分钟内，除CYS-NEM以外，两种试剂均获得了完全衍生化，而CYS-NEM的效率为97％。疏水性更高的NPEM提供了更好的硫醇离子化效果，与NEM相比，GSH的增幅为2.1倍，CYS的增幅为5.7倍。NPEM衍生化导致更广泛的副反应，例如双重衍生化和开环，这阻碍了硫醇浓度的准确测量。NEM和NPEM均显示出CYS衍生物的稳定性差，这是由于其时间依赖性地转化为环半胱氨酸-马来酰亚胺衍生物。两种试剂还显示出与含胺代谢物的显着反应性，具体取决于衍生过程中使用的pH值，但是发现整个NEM对巯基的选择性比NPEM高。考虑到所有评估标准，NEM被选为更合适的巯基保护和衍生试剂，但建议严格控制pH 7.0以最大程度减少副反应。这项工作说明了在硫醇衍生剂评估过程中表征副产物和衍生物稳定性的重要性，并为提高硫醇测定的准确性提供了基础知识。马来酰亚胺衍生化过程中错误的主要来源包括含胺代谢物的衍生化，某些硫醇（CYS和NAC）的衍生物稳定性差，以及副反应，特别是如果未将试剂的开环控制到最小的话。图形概要。