Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke Street West, Montréal, QC, H4B 1R6, Canada.
PERFORM Centre, Concordia University, 7200 Sherbrooke Street West, Montréal, QC, H4B 1R6, Canada.
Anal Bioanal Chem. 2020 Mar;412(7):1639-1652. doi: 10.1007/s00216-020-02398-x. Epub 2020 Feb 4.
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.
巯基还原态和氧化态的比例（主要是谷胱甘肽和氧化型谷胱甘肽）是评估氧化应激的生物标志物之一。巯基浓度的准确测量具有挑战性，因为巯基在样品处理过程中很容易被氧化。衍生化通常用于保护巯基免受氧化。本工作旨在系统比较两种具有细胞通透性的衍生化试剂：N-乙基马来酰亚胺（NEM）和（R）-（+）-N-（1-苯乙基）马来酰亚胺（NPEM），比较其衍生化效率、离子化增强、副产物形成、对巯基的反应选择性、反应 pH 依赖性和衍生物稳定性。所有巯基测量和副产物的表征均采用联苯反相高效液相色谱-高分辨率质谱（LC-HRMS）进行。使用 4 种巯基（半胱氨酸（CYS）、同型半胱氨酸、N-乙酰半胱氨酸（NAC）和谷胱甘肽（GSH））来评价。使用巯基：衍生化试剂 1:10 的比例，除 CYS-NEM 外，两种试剂均在 30 分钟内完成完全衍生化，其中 CYS-NEM 的效率为 97%。疏水性更强的 NPEM 使巯基的离子化效果更好，与 NEM 相比，GSH 的增强范围为 2.1x，CYS 的增强范围为 5.7x。NPEM 衍生化导致更广泛的副反应，如双衍生化和开环，这阻碍了巯基浓度的准确测量。由于其时间依赖性转化为环化半胱氨酸-马来酰亚胺衍生物，CYS 衍生物的两种试剂也表现出较差的稳定性。两种试剂在衍生化过程中使用的 pH 依赖性也表现出与含胺代谢物的显著反应性，但总体而言，NEM 比 NPEM 对巯基基团更具选择性。考虑到所有评价标准，NEM 被选为更适合巯基保护和衍生化的试剂，但建议严格控制 pH 7.0，以最大限度地减少副反应。这项工作说明了在评价巯基衍生化试剂时，对副产物和衍生物稳定性进行表征的重要性，并为提高巯基测定的准确性提供了基本的理解。马来酰亚胺衍生化过程中的主要误差来源包括含胺代谢物的衍生化、某些巯基（CYS 和 NAC）的衍生物稳定性差，以及副反应，特别是如果不最小化试剂的开环。
