Abstract
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 Cys34 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 Cys34. Furthermore, the results of molecular dynamics simulations are indicative that the protein environment around Cys34 allows adduct formation with elongated but not bulky molecules such as Q, and identify important hydrogen bonding interactions and hydrophobic contacts.Graphical abstract
Highlights
Vesicants, chemicals that cause blistering of the skin, have been used as warfare agents for more than 100 years
This norm is laid down in the Chemical Weapons Convention (CWC), which entered into force in 1997, and is implemented by the Organisation for the Prohibition of Chemical Weapons (OPCW) [6]
The most long-lasting in vivo biomarkers of exposure are protein-adducts such as those formed with human serum albumin (HSA)
Summary
Chemicals that cause blistering of the skin, have been used as warfare agents for more than 100 years. Verification of exposure is an important capability in order to guide medical therapy of casualties and for generating evidence that a violation of the international norm on the nonuse of chemical weapons has occurred [5]. HSA-adducts result from covalent linkage of SM to, e.g., the Cys residue, and have emerged as the most prominent biomarkers [7,8,9,10] They have been used on several occasions for the analysis of samples from OPCW interlaboratory exercises and from exposed victims [11,12,13,14]
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