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Identification of the novel leaving group adducts with human albumin expands the basic knowledge on molecular toxicology of the nerve agent VX. Furthermore, the presented μLC/ESI HR MS/MS method might be of relevance for verification of VX poisoning. Copyright © 2016 John Wiley & Sons, Ltd.

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 (μLC-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 in HSA (DPAET-CysPro, DEAET-CysPro). We also identified tripeptide disulfide-adducts containing Cys (MetProCys-DPAET, MetProCys-DEAET) and to a lesser extent Cys (AspIleCys-DPAET, AspIleCys-DEAET). Synthetic tripeptide references were used for confirmation of the postulated structures by μLC-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 μM nerve agent for tyrosine-adducts, 1-3 μM nerve agent for CysPro-adducts, and 6 μM 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 °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. Graphical abstract Modification of human serum albumin (HSA) by V-type nerve agents Chinese VX (CVX) and RussianVX (RVX). Various tyrosine residues (Tyr???)n (e.g. most reactive Tyr411) were phosphonylated and disulfide-adducts were formed between the thiol-containing leaving group 2-(diethylamino)ethanethiol (DEAET) and at least three cysteine residues (Cys34, Cys448 and Cys514). Pronase-mediated proteolysis produced low-molecular cleavage products including phosphonylated tyrosines, dipeptide (Cys34Pro) and tripeptide (MetProCys448, AspIleCys514) disulfide-adducts that were detected by microbore liquid chromatography-electrospray ionization mass spectrometry/high-resolution mass spectrometry (μLC-ESI MS/HR MS).

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 methionine 329 in human serum albumin (HSA) as a novel target of SM forming a HETE-methionyl sulfonium ion.The alkylated tetrapeptide LeuGlyMet 329 (-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 Met 329 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.

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