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The highly blistering sulfur mustard analogue agent T (bis(2-chloroethylthioethyl) ether), also known as O-mustard or oxy-mustard, is a common impurity in military grade sulfur mustard (SM) and a component of mixtures such as "HT" that are still found in old munitions. Together with sesquimustard (Q), it is the most important SM analogue and tightly regulated as a Schedule 1 chemical under the Chemical Weapons Convention. We report the adducts of T with nucleophilic Cys 34 and other residues in human serum albumin (HSA) formed in vitro. A micro liquid chromatography electrospray ionization high-resolution tandem-mass spectrometry method (µLC-ESI MS/HR MS) was developed for the detection and identification of biomarker peptides alkylated by a T-derived hydroxyethylthioethyloxyethylthioethyl (HETEOETE)-moiety (as indicated by an asterisk below). Following proteolysis of T-exposed human plasma with pronase, the dipeptide Cys 34 *Pro and the single amino acid residue His* were produced. The use of proteinase K yielded Cys 34 *ProPhe and the use of pepsin generated ValThrGlu 48 *Phe, AlaGlu 230 *ValSerLysLeu, and LeuGlyMet 329 *Phe. Corresponding peptide-adducts of SM and Q were detected in a common workflow that in principle allowed the estimation of the mustard or mustard composition encountered during exposure. Novel adducts of Q at the Glu 230 and Met 239 residues were detected and are reported accordingly. Based on molecular dynamics simulations, we identified regular interactions of the Cys 34 (-HETEOETE)-moiety with several glutamic acid residues in HSA including Glu 86 , which is not an obvious interaction partner by visual inspection of the HSA crystal structure. The existence of this and other intramolecular cross-links was experimentally proven for the first time., (© 2024. The Author(s).)

Sesquimustard (Q) is a powerful blistering agent that contains additional sulfur atoms. Sulfur mustard causes covalent bonding by alkylating nucleophilic groups of biologically important macromolecules such as lipids, proteins, DNA, or RNA. Most cells maintain relatively high amounts of a unique tripeptide called glutathione (GSH) (γ-glutamyl-cysteinyl glycine), which possesses a free thiol group, to prevent unwanted reactions caused by reactive chemical entities. Moreover, these thiol groups on cysteines (Cys) are the main target for alkylation. Although Q is the most potent vesicant among sulfur mustards, research studies identifying biomarkers of Q are very limited. Therefore, here in this study, we aimed to identify the GSH and Cys conjugates of Q using mass spectrometric methods and to observe the formation of these conjugates in HaCat cell culture following exposure to different doses. We identified four different conjugates of Q, which are bis-glutathionyl ethylthioethylthioethyl conjugate (GSH-ETETE-GSH), hydroxyethylthioethylthioethyl glutathione conjugate (HETETE-GSH), bis-cysteinyl ethylthioethylthioethyl conjugate (Cys-ETETE-Cys), and hydroxyethylthioethylthioethyl cysteine conjugate (HETETE-Cys). The identity of the conjugates was elucidated using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). We also investigated changes in conjugate formation with exposure concentration and time elapsed after exposure in the cell culture. After exposure, GSH conjugates decreased until 1st hour, while Cys conjugates increased until 6th hour. We also observed that conjugate formation depended on the concentration of Q. This is the first study to elucidate the conjugates of Q dependent on GSH conjugation. As biomarkers are essential tools for evaluating exposure to Q, this study contributes to the limited number of studies identifying biomarkers for Q., (© 2024. The Author(s).)

Transient receptor potential (TRP) channels are important in the sensing of pain and other stimuli. They may be triggered by electrophilic agonists after covalent modification of certain cysteine residues. Sulfur mustard (SM) is a banned chemical warfare agent and its reactivity is also based on an electrophilic intermediate. The activation of human TRP ankyrin 1 (hTRPA1) channels by SM has already been documented, however, the mechanism of action is not known in detail. The aim of this work was to purify hTRPA1 channel from overexpressing HEK293 cells for identification of SM-induced alkylation sites. To confirm hTRPA1 isolation, Western blot analysis was performed showing a characteristic double band at 125 kDa. Immunomagnetic separation was carried out using either an anti-His-tag or an anti-hTRPA1 antibody to isolate hTRPA1 from lysates of transfected HEK293 cells. The identity of the channel was confirmed by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry. Following SM exposure, hTRPA1 channel modifications were found at Cys 462 and Cys 665 , as well as at Asp 339 and Glu 341 described herein for the first time. Since Cys 665 is a well-known target of hTRPA1 agonists and is involved in hTRPA1 activation, SM-induced modifications of cysteine, as well as aspartic acid and glutamic acid residues may play a role in hTRPA1 activation. Considering hTRPA1 as a target of other SM-related chemical warfare agents, analogous adducts may be predicted and identified applying the analytical approach described herein., (© 2022. The Author(s).)

In the recent past, the blister agent sulfur mustard (SM) deployed by the terroristic group Islamic State has caused a huge number of civilian and military casualties in armed conflicts in the Middle East. The vaporized or aerolized agent might be inhaled and have direct contact to skin and hair. Reaction products of SM with plasma proteins (adducts) represent well-established systemic targets for the bioanalytical verification of exposure. The SM-derived hydroxyethylthioethyl (HETE)-moiety is attached to nucleophilic amino acid side chains and allows unambiguous adduct detection. For shipping of common blood and plasma samples, extensive packaging rules are to be followed as these matrices are considered as potentially infectious material. In contrast, hair is considered as non-infectious thus making its handling and transportation much less complicated. Therefore, we addressed this matrix to develop a procedure for bioanalytical verification. Following optimized lysis of SM-treated human scalp hair and pepsin-catalyzed proteolysis of adducts of keratin type I and II, microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS) was used to detect three alkylated keratin-derived biomarker peptides: AE(-HETE)IRSDL, FKTIE(-HETE)EL, and LE(-HETE)TKLQF simultaneously. All bear the HETE-moiety bound to a glutamic acid residue. Protein adducts were stable for at least 14 weeks at ambient temperature and contact to air, and were not affected by washing the hair with shampoo. The biomarker peptides were also obtained from beard, armpit, abdominal, and pubic hair. This is the first report introducing stable local peptide adduct biomarkers from hair, that is easily accessible by a non-invasive sampling process., (© 2022. The Author(s).)

The primary aim of this study was to identify biomarkers of exposure to some so-called Schedule 1 sulfur mustard (HD) analogues, in order to facilitate and expedite their retrospective analysis in case of alleged use of such compounds. Since these HD analogues can be regarded as model compounds for possible impurities of HD formed during synthesis processes, the secondary aim was to explore to which extent these biomarkers can be used for chemical provenancing of HD in case biomedical samples are available. While the use of chemical attribution signatures (CAS) for neat chemicals or for environmental samples has been addressed quite frequently, the use of CAS for investigating impurities in biomedical samples has been addressed only scarcely. Human plasma was exposed to each of the five HD analogues. After pronase or proteinase K digestion of precipitated protein and sample work-up, the histidine (His) and tripeptide (CPF) adducts to proteins were analyzed, respectively. Adducts of the analogues could still be unambiguously identified next to the main HD adducts in processed plasma samples after exposure to HD mixed with each of the analogues, at a 1% level relative to HD. In conclusion, we have identified plasma protein adducts of a number of HD analogues, which can be used as biomarkers to assess an exposure to these Schedule 1 chemicals. We have shown that adducts of these analogues can still be analyzed after work-up of plasma samples which had been exposed to these analogues in a mixture with HD, supporting the hypothesis that biomedical sample analysis might be useful for chemical provenancing.

We present the forensic analyses of plasma samples of human victims exposed to sulfur mustard (SM) in a crisis region in the Middle East in 2015. A few hours after exposure, poisoned persons showed typical signs and symptoms of percutaneous SM exposure including erythema and later on blisters and hardly healing skin wounds. Blood samples were collected 15 days after poisoning to be analyzed for the presence of long-lived protein-adduct biomarkers to verify SM poisoning. We applied a novel bioanalytical toolbox targeting four human serum albumin-derived biomarkers that were made accessible after plasma proteolysis. These adducts contained the SM-specific hydroxyethylthioethyl moiety either bound to the thiol group of a cysteine residue (C 34 *) or to the side-chain carboxylic group of a glutamic acid residue (E 230 *). Peptide biomarkers were produced from plasma of the victims using proteinase K (C 34 *PF), pronase (C 34 *P) and pepsin (AE 230 *VSKL and LQQC 34 *PFEDHVKL) for enzymatic protein cleavage. Separation and detection were carried out by selective micro-liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS). In addition to this site-specific adduct detection, a general approach after alkaline hydrolysis of the plasma protein fraction was applied. Liberated thiodiglycol (TDG) was derivatized with heptafluorobutyric anhydride and detected by gas chromatography-electron ionization mass spectrometry (GC-EI MS). The different bioanalytical methods yielded congruent results confirming SM poisoning for all patients who showed clinical signs and symptoms. This is the first time that real cases of SM poisoning were confirmed and presented by such a broad compilation of protein-derived biomarkers.

As part of a program to develop methods for the verification of alleged exposure to sulphur mustard, we synthesized and characterized three amino acid adducts presumably formed by alkylation of haemoglobin: 4-(2-hydroxyethylthioethyl)-L-aspartate, 5-(2-hydroxyethylthioethyl)L-glutamate and N1- and N3-(2-hydroxyethylthioethyl)-L-histidine. Suitable derivatization methods for GC/MS analysis were developed for these adducts as well as for the cysteine and the N-terminal valine adduct. Incubation of human blood with [35S]sulfur mustard in vitro followed by acidic hydrolysis of isolated globin and derivatization with Fmoc-Cl afforded three radioactive peaks upon HPLC analysis, one of which coeluted with the synthetic Fmoc derivative of N1/N3-(2-hydroxyethylthioethyl)-L-histidine. After pronase digestion of globin the adducts of histidine, glutamic acid, aspartic acid, cysteine and N-terminal valine could be tentatively identified and quantitated. Final identification was obtained from GC/MS analysis. The most abundant adduct, N1/N3-(2-hydroxyethylthioethyl)-L-histidine, could not be sensitively analysed by GC/MS. A convenient LC-tandem MS procedure was developed for this compound, enabling the detection of exposure of human blood to 10 microM sulphur mustard in vitro.