Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany.
Department of Bioengineering Sciences, Weihenstephan-Triesdorf University of Applied Sciences, Freising, Germany.
Drug Test Anal. 2023 Jul;15(7):730-744. doi: 10.1002/dta.3459. Epub 2023 Feb 23.
We herein present for the first time a micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS) procedure to detect phosphonylated tyrosine (Tyr) and lysine (Lys) residues obtained from human hair exposed to organophosphorus nerve agents (OPNA). In general, toxic OPNA react with endogenous blood proteins causing the formation of adducts representing well-known targets for biomedical analysis to prove exposure. In contrast, no protein-derived biomarker has been introduced so far to document local exposure of hair. Accordingly, we developed and characterized a μLC-ESI MS/HR MS method for the analysis of scalp hair exposed to OPNA in vitro. Type I and Type II keratin from hair was dissolved during lysis, precipitated and subjected to pronase-catalyzed hydrolysis yielding single adducted Lys and in a much higher amount Tyr residues. Exposure to sarin caused the adduction of an isopropyl methylphosphonic acid moiety and exposure to VX yielded adducts of ethyl methylphosphonic acid, well suited as biomarkers of exposure. These were of appropriate stability in the autosampler for 24 h. The biomarker yield obtained from hair of six individuals as well as from hair of six different parts of the body of one individual (armpit, beard, leg, arm, scalp, and pubic) differed reasonably indicating the variable individual protein composition and structure of hair. Exposed hair stored at ambient temperature for 9 weeks with contact to air and daylight showed stability of all adducts and therefore their suitability for verification of exposure.
我们首次提出了一种微液相色谱-电喷雾电离高分辨串联质谱（μLC-ESI MS/HR MS）程序，用于检测暴露于有机磷神经毒剂（OPNA）的人发中磷酸化酪氨酸（Tyr）和赖氨酸（Lys）残基。一般来说，有毒的 OPNA 与内源性血液蛋白反应，形成加合物，这些加合物是生物医学分析中证明暴露的众所周知的靶标。相比之下，迄今为止，尚未引入任何源自蛋白质的生物标志物来记录头发的局部暴露。因此，我们开发并表征了一种用于分析体外暴露于 OPNA 的头皮毛发的 μLC-ESI MS/HR MS 方法。在裂解过程中溶解 I 型和 II 型角蛋白，沉淀并进行胰蛋白酶催化水解，得到单加合 Lys 和更多量的 Tyr 残基。沙林暴露导致异丙基甲基膦酸部分的加合，VX 暴露导致乙基甲基膦酸的加合，非常适合作为暴露的生物标志物。这些在自动进样器中 24 小时内具有适当的稳定性。从六个人的头发中以及从一个人身体的六个不同部位（腋窝、胡须、腿部、手臂、头皮和阴部）的头发中获得的生物标志物产量差异合理，表明个体蛋白质组成和头发结构的差异。暴露的头发在室温下储存 9 周，与空气和日光接触，所有加合物均稳定，因此适合用于验证暴露。
