Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstr. 11, 80937, Munich, Germany.
Walther-Straub-Institut, Ludwig-Maximilians-Universität, Munich, Germany.
Arch Toxicol. 2023 Jul;97(7):1873-1885. doi: 10.1007/s00204-023-03521-4. Epub 2023 Jun 1.
VX is a highly toxic organophosphorus nerve agent that reacts with a variety of endogenous proteins such as serum albumin under formation of adducts that can be targeted by analytical methods for biomedical verification of exposure. Albumin is phosphonylated by the ethyl methylphosphonic acid moiety (EMP) of VX at various tyrosine residues. Additionally, the released leaving group of VX, 2-(diisopropylamino)ethanethiol (DPAET), may react with cysteine residues in diverse proteins. We developed and validated a microbore liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS) method enabling simultaneous detection of three albumin-derived biomarkers for the analysis of rat plasma. After pronase-catalyzed cleavage of rat plasma proteins single phosphonylated tyrosine residues (Tyr-EMP), the Cys(-DPAET)Pro dipeptide as well as the rat-specific LeuProCys(-DPAET) tripeptide were obtained. The time-dependent adduct formation in rat plasma was investigated in vitro and biomarker formation during proteolysis was optimized. Biomarkers were shown to be stable for a minimum of four freeze-and-thaw cycles and for at least 24 h in the autosampler at 15 °C thus making the adducts highly suited for bioanalysis. Cys(-DPAET)Pro was superior compared to the other serum biomarkers considering the limit of identification and stability in plasma at 37 °C. For the first time, Cys(-DPAET)Pro was detected in in vivo specimens showing a time-dependent concentration increase after subcutaneous exposure of rats underlining the benefit of the dipeptide disulfide biomarker for sensitive analysis.
VX 是一种高毒性有机磷神经毒剂，在形成加合物的过程中与各种内源性蛋白质（如血清白蛋白）反应，这些加合物可以通过分析方法来检测生物医学暴露。VX 的乙基甲基膦酸部分（EMP）在各种酪氨酸残基上将白蛋白膦酰化。此外，VX 的释放离去基团 2-（二异丙基氨基）乙硫醇（DPAET）可能与各种蛋白质中的半胱氨酸残基反应。我们开发并验证了一种微流液相色谱-电喷雾电离高分辨串联质谱（µLC-ESI MS/HR MS）方法，能够同时检测三种白蛋白衍生的生物标志物，用于分析大鼠血浆。在蛋白酶催化大鼠血浆蛋白裂解后，获得了单膦化酪氨酸残基（Tyr-EMP）、Cys（-DPAET）Pro 二肽以及大鼠特异性 LeuProCys（-DPAET）三肽。在体外研究了大鼠血浆中加合物的时变形成，优化了蛋白水解过程中生物标志物的形成。研究表明，生物标志物在至少四个冻融循环和至少 24 小时的自动进样器中在 15°C 下稳定，因此非常适合生物分析。与其他血清生物标志物相比，Cys（-DPAET）Pro 考虑到 37°C 下的鉴定限和稳定性更具优势。首次在体内标本中检测到 Cys（-DPAET）Pro，表明大鼠皮下暴露后其浓度随时间呈依赖性增加，这突显了二肽二硫生物标志物在敏感分析中的优势。
