State Key Laboratory of NBC Protection for Civilian, Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing, 102205, People's Republic of China.
School of Chemistry and Chemical Engineering, Nanjing University of Sciences & Technology, Nanjing, 210094, People's Republic of China.
Anal Bioanal Chem. 2022 Jun;414(14):4179-4188. doi: 10.1007/s00216-022-04070-y. Epub 2022 Apr 27.
A major challenge for the unequivocal verification of alleged exposure to sulfur mustard (HD) lies in identifying its multiple modifications on endogenous proteins and utilizing these modified proteins to achieve accurate, sensitive, and rapid detection for retrospective analysis of HD exposure. As the most abundant protein in human plasma, human serum albumin (HSA) can react with many xenobiotics, such as HD, to protect the body from damage. The HSA adducts induced by HD have been used as biomarkers for the verification of HD exposure. In this study, the modification sites on HSA by HD were identified through application of the bottom-up strategy used in proteomics, and 41 modified sites were discovered with seven types of amino acids, of which 3 types were not previously reported. Then, different enzymes, including pepsin, endoproteinase Glu-C, and pronase, were applied to digest HD-HSA to produce adducts with hydroxyethylthioethyl (HETE) groups, which may be used as potential biomarkers for HD exposure. As candidates for retrospective analysis, sixteen adducts were obtained and characterized with ultra-high-pressure liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry (UHPLC-QE Focus MS). These potential biomarkers were evaluated in human plasma that was exposed in vitro to HD and five of its analogues. This study integrated the identification of modification sites through application of the bottom-up strategy of proteomics and screening biomarkers, providing a novel strategy for retrospective detection of the exposure of xenobiotic chemicals.
鉴定疑似硫芥（HD）暴露的一个主要挑战在于识别内源性蛋白质上的多种修饰，并利用这些修饰蛋白质来实现准确、敏感和快速的检测，以便对 HD 暴露进行回顾性分析。人血清白蛋白（HSA）作为人血浆中最丰富的蛋白质，可与许多外源化学物（如 HD）反应，以保护身体免受损伤。HD 诱导的 HSA 加合物已被用作验证 HD 暴露的生物标志物。在这项研究中，通过应用蛋白质组学中的自下而上策略鉴定了 HSA 上的 HD 修饰位点，发现了 41 个具有七种类型氨基酸的修饰位点，其中 3 种类型以前未报道过。然后，应用不同的酶，包括胃蛋白酶、内切蛋白酶 Glu-C 和糜蛋白酶，消化 HD-HSA 以产生具有羟乙基硫乙基（HETE）基团的加合物，这些加合物可能可作为 HD 暴露的潜在生物标志物。作为回顾性分析的候选物，获得并通过超高效液相色谱与四极杆轨道阱质谱（UHPLC-QE Focus MS）对 16 个加合物进行了表征。在体外暴露于 HD 和其五种类似物的人血浆中评估了这些潜在的生物标志物。本研究通过应用蛋白质组学的自下而上策略和筛选生物标志物，整合了修饰位点的鉴定，为外源性化学物质暴露的回顾性检测提供了一种新策略。
