van 't Hoff Institute for Molecular Sciences, Faculty of Science, University of Amsterdam, P.O. Box 94157, Amsterdam 1090GD, The Netherlands.
TNO Defence, Safety and Security, Dep. CBRN Protection, Lange Kleiweg 137, Rijswijk 2288GJ, The Netherlands.
Chem Res Toxicol. 2022 Jun 20;35(6):1070-1079. doi: 10.1021/acs.chemrestox.2c00053. Epub 2022 May 27.
Chlorine is a widely available industrial chemical and involved in a substantial number of cases of poisoning. It has also been used as a chemical warfare agent in military conflicts. To enable forensic verification, the persistent biomarkers 3-chlorotyrosine and 3,5-dichlorotyrosine in biomedical samples could be detected. An important shortfall of these biomarkers, however, is the relatively high incidence of elevated levels of chlorinated tyrosine residues in individuals with inflammatory diseases who have not been exposed to chlorine. Therefore, more reliable biomarkers are necessary to distinguish between endogenous formation and exogeneous exposure. The present study aims to develop a novel diagnostic tool for identifying site-specific chlorinated peptides as a more unambiguous indicator of exogeneous chlorine exposure. Human blood plasma was exposed in vitro to various chlorine concentrations, and the plasma proteins were subsequently digested by pronase, trypsin, or pepsin. After sample preparation, the digests were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). In line with other studies, low levels of 3-chlorotyrosine and 3,5-dichlorotyrosine were found in blank plasma samples in this study. Therefore, 50 site-specific biomarkers were identified, which could be used as more unambiguous biomarkers for chlorine exposure. Chlorination of the peptides TYETTLEK, YKPGQTVK, YQQKPGQAPR, HYEGSTVPEK, and YLYEIAR could already be detected at moderate in vitro chlorine exposure levels. In addition, the latter two peptides were found to have dichlorinated fragments. Especially, YLYEIAR, with a distinct chlorination pattern in the MS spectra, could potentially be used to differentiate exogeneous exposure from endogenous causes as other studies reported that this part of human serum albumin is nitrated rather than chlorinated under physiological conditions. In conclusion, trypsin digestion combined with high-resolution MS analysis of chlorinated peptides could constitute a valuable technique for the forensic verification of exposure to chlorine.
氯是一种广泛存在的工业化学品，涉及大量中毒案例。它也曾在军事冲突中被用作化学战剂。为了进行法医验证，可以检测生物医学样本中持久的生物标志物 3-氯酪氨酸和 3,5-二氯酪氨酸。然而，这些生物标志物的一个重要缺陷是，在没有接触过氯的患有炎症性疾病的个体中，氯化酪氨酸残基的水平升高的发生率相对较高。因此，需要更可靠的生物标志物来区分内源性形成和外源性暴露。本研究旨在开发一种新的诊断工具，以识别特定部位的氯化肽作为外源性氯暴露的更明确指标。体外将人血浆暴露于不同的氯浓度下，然后用蛋白酶、胰蛋白酶或胃蛋白酶消化血浆蛋白。在样品制备后，通过液相色谱串联质谱（LC-MS/MS）和液相色谱高分辨率串联质谱（LC-HRMS/MS）分析提取物。与其他研究一致，本研究中在空白血浆样本中也发现了低水平的 3-氯酪氨酸和 3,5-二氯酪氨酸。因此，鉴定了 50 种特定部位的生物标志物，它们可用作更明确的氯暴露生物标志物。在中等体外氯暴露水平下，已经可以检测到肽 TYETTLEK、YKPGQTVK、YQQKPGQAPR、HYEGSTVPEK 和 YLYEIAR 的氯化。此外，后两种肽被发现具有二氯化片段。特别是 YLYEIAR，其 MS 光谱中的氯化图案明显，可能有助于将外源性暴露与内源性原因区分开来，因为其他研究报告说，在生理条件下，人血清白蛋白的这一部分被硝化而不是氯化。总之，用高分辨率 MS 分析对氯肽进行胰蛋白酶消化可以构成一种有价值的技术，用于法医验证氯暴露。
