Bioanalytical verification of V-type nerve agent exposure: simultaneous detection of phosphonylated tyrosines and cysteine-containing disulfide-adducts derived from human albumin
Bioanalytical verification of V-type nerve agent exposure: simultaneous detection of phosphonylated tyrosines and cysteine-containing disulfide-adducts derived from human albumin
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DOI：
10.1007/s00216-017-0787-7
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发表时间：
2018-02-01
影响因子：
4.3
通讯作者：
John, Harald
中科院分区：
文献类型：
--
作者：
Kranawetvogl, Andreas;Kueppers, Jim;John, Harald
神经毒剂仍然对文职和军事人员构成严重威胁，中东的暴力冲突就证明了这一点。为了验证中毒，与内源性蛋白质（例如人血清白蛋白、HSA）的共价加合物是有价值的长期生物标志物。因此，我们开发了一种微孔液相色谱-电喷雾电离质谱/高分辨率质谱（μ LC-ESI MS/HR MS）方法，用于同时检测 V 型神经毒剂 VX、中国 VX（CVX）和俄罗斯 VX（RVX）的 HSA 加合物。链霉蛋白酶催化的蛋白水解后，除了磷酸化酪氨酸残基之外，还检测到新的二硫键加合物。二肽二硫化物加合物在 V 型神经毒剂的含硫醇离去基团（对于 VX 为 2-（二异丙氨基）乙硫醇，DPAET，对于 CVX 和 RVX 为 2-（二乙氨基）乙硫醇，DEAET）和 HSA 中 Cys（34）的游离硫醇基团（DPAET-CysPro，DEAET-CysPro）之间形成。我们还鉴定了含有 Cys(448) (MetProCys-DPAET、MetProCys-DEAET) 和少量 Cys(514) (AspIleCys-DPAET、AspIleCys-DEAET) 的三肽二硫化物加合物。合成三肽参考用于通过 mu LC-ESI MS/HR MS 确认假定的结构。在人血浆中确定了检测下限，三种 V 型神经毒剂几乎相同，对应于酪氨酸加合物的 1-6 μM 神经毒剂、CysPro 加合物的 1-3 μM 神经毒剂和 MetProCys 加合物的 6 μM 神经毒剂，从而涵盖了毒理学相关的浓度。蛋白水解动力学的表征表明，所有加合物在 37°C 下 60 至 90 分钟内均达到稳定平台。通过同时监测 V 型神经毒剂、其离去基团和相应的二硫键二聚体来表征加合物形成动力学。此外，研究了加合物形成模式作为HSA与V型神经毒剂摩尔比的函数。
Nerve agents still represent a serious threat to civilian and military personnel as demonstrated by the violent conflict in the Middle East. For verification of poisoning, covalent adducts with endogenous proteins (e.g., human serum albumin, HSA) are valuable long-term biomarkers. Accordingly, we developed a microbore liquid chromatography-electrospray ionization mass spectrometry/high-resolution mass spectrometry (mu LC-ESI MS/HR MS) method for simultaneous detection of HSA-adducts with the V-type nerve agents VX, Chinese VX (CVX), and Russian VX (RVX). Following Pronase-catalyzed proteolysis, novel disulfide-adducts were detected in addition to phosphonylated tyrosine residues. Dipeptide disulfide-adducts were formed between the thiol-containing leaving group of the V-type nerve agents (2-(diisopropylamino)ethanethiol, DPAET, for VX and 2-(diethylamino)ethanethiol, DEAET, for CVX and RVX) and the free thiol group of Cys(34) in HSA (DPAET-CysPro, DEAET-CysPro). We also identified tripeptide disulfide-adducts containing Cys(448) (MetProCys-DPAET, MetProCys-DEAET) and to a lesser extent Cys(514) (AspIleCys-DPAET, AspIleCys-DEAET). Synthetic tripeptide references were used for confirmation of the postulated structures by mu LC-ESI MS/HR MS. Lower limits of detection were determined in human plasma, being nearly identical for the three V-type nerve agents, and corresponded to 1-6 mu M nerve agent for tyrosine-adducts, 1-3 mu M nerve agent for CysPro-adducts, and 6 mu M nerve agent for MetProCys-adducts, thus covering concentrations of toxicological relevance. Characterization of proteolysis kinetics revealed stable plateaus for all adducts being reached between 60 and 90 min at 37 A degrees C. Adduct formation kinetics were characterized by simultaneously monitoring the V-type nerve agent, its leaving group, and the corresponding disulfide dimer. Furthermore, adduct formation patterns were investigated as a function of the molar ratio of HSA to V-type nerve agent.