Graduate Institute of Integrated Medicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung, 40402, Taiwan, ROC; Department of Medical Research, China Medical University Hospital, Taichung, 40447, Taiwan, ROC.
Graduate Institute of Biochemical Sciences, National Taiwan University, No.1, Section 4, Roosevelt Road, Taipei, 106, Taiwan, ROC.
J Chromatogr A. 2018 Jun 1;1552:43-52. doi: 10.1016/j.chroma.2018.04.008. Epub 2018 Apr 5.
Hydrogen sulfide (HS), previously known as a toxic gas, is now recognized as a gasotransmitter along with nitric oxide and carbon monoxide. However, only few methods are available for quantitative determination of HS in biological samples. 2-Iodoacetanilide (2-IAN), a thiol-reacting agent, has been used to tag the reduced cysteine residues of proteins for quantitative proteomics and for detection of cysteine oxidation modification. In this article, we proposed a new method for quantitative analyses of HS and thiol metabolites using the procedure of pre-column 2-IAN derivatization coupled with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). C-Labeled and label-free 2-IAN efficiently react with HS and thiol compounds at pH 9.5 and 65 °C. The derivatives exhibit excellent stability at alkaline conditions, high resolution on reverse phase liquid chromatography and great sensitivity for ESI-MS detection. The measurement of HS, l-cysteine, glutathione, and DL-homocysteine derivatives was validated using C-labeled standard in LC-ESI-MS analyses and exhibited 10 nM-1 μM linear ranges for DL-homocysteine and glutathione and 1 nM-1 μM linear ranges for l-cysteine and HS. In addition, the sequence of derivatization and extraction of metabolites is important in the quantification of thiol metabolites suggesting the presence of matrix effects. Most importantly, labeling with 2-IAN and C-2-IAN isotopologues could achieve quantitative and matched sample comparative analyses with minimal bias using our extraction and labeling procedures before LC-MS analysis.
硫化氢（HS）曾被认为是一种有毒气体，现在与一氧化氮和一氧化碳一起被认为是一种气体递质。然而，目前只有少数方法可用于定量测定生物样品中的 HS。2-碘乙酰胺（2-IAN）是一种巯基反应试剂，已被用于标记蛋白质的还原半胱氨酸残基，用于定量蛋白质组学和检测半胱氨酸氧化修饰。在本文中，我们提出了一种新的方法，用于使用柱前 2-IAN 衍生化与液相色谱-电喷雾电离质谱（LC-ESI-MS）联用，对 HS 和巯基代谢物进行定量分析。C 标记和无标记的 2-IAN 在 pH 9.5 和 65°C 时可有效地与 HS 和巯基化合物反应。衍生物在碱性条件下具有极好的稳定性、反相液相色谱中的高分辨率和 ESI-MS 检测的高灵敏度。使用 LC-ESI-MS 分析中的 C 标记标准对 HS、l-半胱氨酸、谷胱甘肽和 DL-高半胱氨酸衍生物的测定进行了验证，DL-高半胱氨酸和谷胱甘肽的线性范围为 10 nM-1 μM，l-半胱氨酸和 HS 的线性范围为 1 nM-1 μM。此外，代谢物衍生化和提取的顺序对巯基代谢物的定量很重要，这表明存在基质效应。最重要的是，使用 2-IAN 和 C-2-IAN 同位素标记物，并在进行 LC-MS 分析之前采用我们的提取和标记程序，可以实现定量和匹配样品的比较分析，且偏差最小。
