Blum Marc-Michael, Richter Annika, Siegert Markus, Thiermann Horst, John Harald
Blum - Scientific Services, Björnsonweg 70d, 22587, Hamburg, Germany.
Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Straße 2, 12489, Berlin, Germany.
Anal Bioanal Chem. 2020 Nov;412(28):7723-7737. doi: 10.1007/s00216-020-02917-w. Epub 2020 Sep 9.
Apart from the well-known sulfur mustard (SM), additional sulfur-containing blistering chemical warfare agents exist. Sesquimustard (Q) is one of them and five times more blistering than SM. It is a common impurity in mustard mixtures and regularly found in old munitions but can also be used in pure form. Compared to the extensive literature on SM, very little experimental data is available on Q and no protein biomarkers of exposure have been reported. We herein report for the first time the adduct of Q with the nucleophilic Cys residue of human serum albumin (HSA) formed in vitro and introduce two novel bioanalytical procedures for detection. After proteolysis of this HSA adduct catalyzed either by pronase or by proteinase K, two biomarkers were identified by high-resolution tandem mass spectrometry (MS/HR MS), namely a dipeptide and a tripeptide, both alkylated at their Cys residue, which we refer to as HETETE-CP and HETETE-CPF. HETETE represents the Q-derived thio-alkyl moiety bearing a terminal hydroxyl group: "hydroxyethylthioethylthioethyl." Targeting both peptide markers from plasma, a micro liquid chromatography-electrospray ionization tandem mass spectrometry method working in the selected reaction monitoring mode (μLC-ESI MS/MS SRM) was developed and validated as well suited for the verification of exposure to Q. Fulfilling the quality criteria defined by the Organisation for the Prohibition of Chemical Weapons, the novel methods enable the detection of exposure to Q alone or in mixtures with SM. We further report on the relative reactivity of Q compared to SM. Based on experiments making use of partially deuterated Q as the alkylating agent, we rule out a major role for six-membered ring sulfonium ions as relevant reactive species in the alkylation of Cys. Furthermore, the results of molecular dynamics simulations are indicative that the protein environment around Cys allows adduct formation with elongated but not bulky molecules such as Q, and identify important hydrogen bonding interactions and hydrophobic contacts. Graphical abstract.
除了广为人知的硫芥气（SM）外，还存在其他含硫的糜烂性化学战剂。倍半芥子气（Q）就是其中之一，其糜烂性比硫芥气强五倍。它是芥子气混合物中的常见杂质，经常在旧弹药中发现，但也可以以纯形式使用。与关于硫芥气的大量文献相比，关于倍半芥子气的实验数据非常少，并且尚未报道过暴露的蛋白质生物标志物。我们在此首次报告了体外形成的倍半芥子气与人血清白蛋白（HSA）亲核半胱氨酸残基的加合物，并介绍了两种用于检测的新型生物分析方法。在链霉蛋白酶或蛋白酶K催化该HSA加合物的蛋白水解后，通过高分辨率串联质谱（MS/HR MS）鉴定出两种生物标志物，即二肽和三肽，它们的半胱氨酸残基均被烷基化，我们将其称为HETETE-CP和HETETE-CPF。HETETE代表带有末端羟基的倍半芥子气衍生的硫代烷基部分：“羟乙硫基乙硫基乙硫基”。针对血浆中的两种肽标记物，开发并验证了一种在选择反应监测模式下工作的微液相色谱-电喷雾电离串联质谱方法（μLC-ESI MS/MS SRM），该方法非常适合用于验证是否暴露于倍半芥子气。这些新方法符合禁止化学武器组织定义的质量标准，能够检测单独暴露于倍半芥子气或与硫芥气混合暴露的情况。我们还报告了倍半芥子气与硫芥气相比的相对反应活性。基于使用部分氘代倍半芥子气作为烷基化剂的实验，我们排除了六元环硫鎓离子作为半胱氨酸烷基化中相关反应物种的主要作用。此外，分子动力学模拟结果表明，半胱氨酸周围的蛋白质环境允许与诸如倍半芥子气这样的细长但不庞大的分子形成加合物，并确定了重要的氢键相互作用和疏水接触。图形摘要。
