Markus Siegert

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.

Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys²⁸³) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys²⁸³ is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys²⁸³ and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys²⁸³ but was conclusive for the formation of Met⁷⁰-HETE and Met¹⁷⁹-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met⁷⁰Ala, Met¹⁷⁹Leu, and Met⁷⁰Ala/Met¹⁷⁹Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met⁷⁰ and Met¹⁷⁹ residues for CK activity.

Sulfur mustard (SM) is a chemical warfare agent which use is banned under international law and that has been used recently in Northern Iraq and Syria by the so-called Islamic State. SM induces the alkylation of endogenous proteins like albumin and hemoglobin thus forming covalent adducts that are targeted by bioanalytical methods for the verification of systemic poisoning. We herein report a novel biomarker, namely creatine kinase (CK) B-type, suitable as a local biomarker for SM exposure on the skin. Human and rat skin were proven to contain CK B-type by Western blot analysis. Following exposure to SM ex vivo, the CK-adduct was extracted from homogenates by immunomagnetic separation and proteolyzed afterwards. The cysteine residue Cys²⁸² was found to be alkylated by the SM-specific hydroxyethylthioethyl (HETE)-moiety detected as the biomarker tetrapeptide TC(-HETE)PS. A selective and sensitive micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HRMS) method was developed to monitor local CK-adducts in an in vivo study with rats percutaneously exposed to SM. CK-adduct formation was compared to already established DNA- and systemic albumin biomarkers. CK- and DNA-adducts were successfully detected in biopsies of exposed rat skin as well as albumin-adducts in plasma. Relative biomarker concentrations make the CK-adduct highly appropriate as a local dermal biomarker. In summary, CK or rather Cys²⁸² in CK B-type was identified as a new, additional dermal target of local SM exposures. To our knowledge, it is also the first time that HETE-albumin adducts, and HETE-DNA adducts were monitored simultaneously in an in vivo animal study.

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.

Sulfur mustard (SM) is a toxic chemical warfare agent deployed in several conflicts within the last 100 years and still represents a threat in terroristic attacks and warfare. SM research focuses on understanding the pathophysiology of SM and identifying novel biomarkers of exposure. SM is known to alkylate nucleophilic moieties of endogenous proteins, for example, free thiol groups of cysteine residues. The two‐dimensional‐thiol‐differences in gel electrophoresis (2D‐thiol‐DIGE) technique is an initial proteomics approach to detect proteins with free cysteine residues. These amino acids are selectively labeled with infrared‐maleimide dyes visualized after GE. Cysteine residues derivatized by alkylating agents are no longer accessible for the maleimide–thiol coupling resulting in the loss of the fluorescent signal of the corresponding protein. To prove the applicability of 2D‐thiol‐DIGE, this technology was exemplarily applied to neat human serum albumin treated with SM, to lysates from human cell culture exposed to SM as well as to human plasma exposed to CEES (chloroethyl ethyl sulfide, an SM analogue). Exemplarily, the most prominent proteins modified by SM were identified by matrix‐assisted laser desorption/ionization time‐of‐flight (tandem) mass spectrometry, MALDI‐TOF MS(/MS), as creatine kinase (CK) from human cells and as alpha‐1 antitrypsin (A1AT) from plasma samples. Peptides containing the residue Cys²⁸² of CK and Cys²³² of A1AT were unambiguously identified by micro liquid chromatography‐electrospray ionization high‐resolution tandem‐mass spectrometry (μLC‐ESI MS/HR MS) as being alkylated by SM bearing the specific hydroxyethylthioethyl‐(HETE)‐moiety. Both peptides might represent potential biomarkers of SM exposure. This is the first report introducing these endogenous proteins as targets of SM alkylation.

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 (C³⁴*) or to the side-chain carboxylic group of a glutamic acid residue (E²³⁰*). Peptide biomarkers were produced from plasma of the victims using proteinase K (C³⁴*PF), pronase (C³⁴*P) and pepsin (AE²³⁰*VSKL and LQQC³⁴*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.

Exposure to the vesicant sulfur mustard (SM) may lead to erythema and blistering. Toxicity of SM is hypothesized due to the alkylation of DNA bases and nucleophilic amino acid side chains in proteins (adducts) by forming the hydroxyethylthioethyl (HETE) moiety. Despite its prohibition by the chemical weapons convention, SM still represents a serious threat to military personnel and civilians. Therefore, development and improvement of forensic analytical methods for the verification of SM exposure is of high interest. Protein adducts have been shown to be highly suitable and beneficial biomarkers of poisoning. Herein we present methionine³²⁹ in human serum albumin (HSA) as a novel target of SM forming a HETE‐methionyl sulfonium ion. The alkylated tetrapeptide LeuGlyMet³²⁹(‐HETE)Phe, LGM(‐HETE)F, was detected after pepsin‐mediated proteolysis and subsequent analysis by microbore liquid chromatography–electrospray ionization–high‐resolution tandem‐mass spectrometry. Compound identity was confirmed by a synthetic reference. Proteolysis conditions for HSA were optimized towards maximum yield of LGM(‐HETE)F and its limit of identification (32.3 nM SM in serum) was similar to those of the established HSA‐derived biomarkers HETE‐CysPro and HETE‐CysProPhe (15.6 nM SM in serum). Stability of the alkylated Met³²⁹ in vitro and in vivo was limited to 5 days making this modification a beneficial short‐time biomarker. Furthermore, it was found that the HETE‐methionyl sulfonium ion can transfer its HETE moiety to the side chain of cysteine and glutamic acid as well as to the N‐terminus of peptides and proteins in vitro thus revealing novel insights into the molecular toxicity of SM.

Rationale: Dimethoate (DIM, S=P(OMe) ₂ ‐S‐CH ₂ ‐C(O)‐NH‐CH ₃ ) is a dimethyl phosphorodithioate pesticide widely used in agri‐ and horticulture that undergoes biotransformation in vivo by desulfuration into its more toxic oxono‐derivative omethoate (OM, O=P(OMe) ₂ ‐S‐CH ₂ ‐C(O)‐NH‐CH ₃ ). OM inhibits acetylcholinesterase thus provoking cholinergic crisis in vivo , ultimately leading to death. Quantitative approaches for the determination of DIM and OM in environmental and toxicological samples make use of tandem mass spectrometry (MS ² ). Nevertheless, so far interpretation of resulting product ions is incomplete and sometimes contradictory. Methods: DIM and OM as well as their deuterated analogues (fully deuterated at both methoxy groups bound to the phosphorus atom) were analyzed by MS ² and MS ³ after positive electrospray ionization and collision‐induced dissociation (CID) in a linear ion trap to characterize fragmentations. The accurate masses of product ions were determined in a time‐of‐flight mass analyzer. Hydrogen/deuterium (H/D)‐exchange experiments were carried out for further support of product ion identification. In addition, density functional theory (DFT) computations were used to calculate both the most stable protonation sites of DIM and OM and the changes in the diverse bond lengths after protonation. Results: Some identical and some related product ions of DIM and OM were found but also striking individual differences. Fragmentation pathways were proposed and product ions identified. Most fragmentations followed the common rules of charge migration fragmentation. DFT calculations supported experimental findings. Conclusions: Discrepancies present in the literature so far are clarified and a deeper insight is provided into the fragmentation processes of organophosphorus pesticides. The combination of diverse experimental and theoretical approaches yielded consistent results, thus demonstrating continuous progress in understanding gas‐phase reactions in MS experiments.