Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, PR China; Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, PR China.
School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, 210023, PR China; Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, PR China.
Anal Chim Acta. 2021 Nov 1;1184:339016. doi: 10.1016/j.aca.2021.339016. Epub 2021 Sep 1.
Sulfur-containing metabolites are related to several physiologic disorders and metabolic diseases. In this study, a simultaneous identification and quantification strategy in one batch for determination of sulfhydryl-containing metabolites was developed using stable isotope labeling combined with liquid chromatography-tandem mass spectrometry (SIL-LC-MS). In the proposed method, a pair of isotope labeling reagents, D/D-N-ethylmaleimide (D/D-NEM), was used to derivatize sulfhydryl-containing metabolites in blood and plasma of normal- and high-fat-diet (NFD and HFD) hamsters for reduced (-SH) and total (-SH, -S-S-, S-glutathionylated proteins) analysis. Quality control (QC) samples and test samples were prepared for LC-MS analysis. First, both QC samples and stable isotope labeled internal standards were used to monitor the status of the instrument and ensure the reliability of the analysis. Subsequently, an inhouse database containing 45 sulfhydryl-containing metabolites was established by MS based on QC samples. Then, qualitatively differential sulfhydryl-containing metabolites were found by MS between the NFD and HFD hamsters of the test samples, including 3 in reduced and 8 in total analysis of blood samples, and 2 in reduced and 2 in total analysis of plasma samples. Next, in quantitative analysis, satisfied linearities for 6 sulfhydryl-containing metabolites were obtained with the correlation coefficient (R) > 0.99 and absolute quantification was carried out. The results showed that glutathione and cysteine have different concentrations in blood and plasma of hamsters. Finally, the correlation of sulfhydryl-containing metabolites with blood lipid and oxidative stress levels was determined, which provided insight into the hyperlipidemia-related oxidative stress. Taken together, the developed method of simultaneous identification with the inhouse database and MS and quantification with standards in one batch provides a promising strategy for the analysis of sulfhydryl-containing metabolites in biological samples, which may promote the in-depth investigation on sulfhydryl-containing metabolites and related diseases.
含硫代谢物与多种生理紊乱和代谢性疾病有关。在这项研究中，我们开发了一种同时鉴定和定量的策略，即使用稳定同位素标记结合液相色谱-串联质谱（SIL-LC-MS），在一批样品中同时测定含巯基代谢物。在所提出的方法中，我们使用一对同位素标记试剂 D/D-N-乙基马来酰亚胺（D/D-NEM），对正常饮食（NFD）和高脂饮食（HFD）仓鼠的血液和血浆中的含巯基代谢物进行衍生化，用于还原（-SH）和总巯基（-SH、-S-S-、S-谷胱甘肽化蛋白）分析。我们制备了 LC-MS 分析用的质控（QC）样品和测试样品。首先，QC 样品和稳定同位素标记的内标都用于监测仪器状态，以确保分析的可靠性。随后，我们通过 MS 基于 QC 样品建立了一个包含 45 种含巯基代谢物的内部数据库。然后，通过对测试样品中 NFD 和 HFD 仓鼠之间的 MS 比较，发现了 3 种在还原分析中、8 种在总巯基分析中存在差异的含巯基代谢物，在还原和总分析中分别在血浆样品中发现了 2 种。接下来，在定量分析中，6 种含巯基代谢物均获得了满意的线性关系（R>0.99），并进行了绝对定量。结果表明，谷胱甘肽和半胱氨酸在仓鼠的血液和血浆中的浓度不同。最后，确定了含巯基代谢物与血脂和氧化应激水平的相关性，这为高血脂相关氧化应激提供了新的认识。总之，本研究开发的同时鉴定方法结合内部数据库和 MS，以及在一批样品中使用标准进行定量的方法，为生物样品中含巯基代谢物的分析提供了一种很有前途的策略，可能会促进对含巯基代谢物及其相关疾病的深入研究。
