Alkylated glutamic acid and histidine derived from protein‐adducts indicate exposure to sulfur mustard in avian serum

Abstract

Sulfur mustard (SM, bis[2-chloroethyl]-sulfide) is a banned chemical warfare agent deployed in the violent conflict in the Middle East poisoning humans and animals.For legal reasons, bioanalytical methods are mandatory proving exposure to SM. Reaction products (adducts) of SM with endogenous proteins, for example, serum albumin (SA), are valuable long-lived targets for analysis. Whereas nearly all methods known so far focus on human proteins, we address for the first time neat chicken SA and avian serum from chicken, duck, and ostrich. After proteolysis, protein precipitation, evaporation of the supernatant, and re-dissolution analysis were performed by micro-liquid chromatography-electrospray ionization tandem-mass spectrometry in the selected reaction monitoring mode, μLC-ESI MS/MS (SRM), for detection of the hydroxyethylthioethyl product ion [HETE] + at m/z 105.0. After in vitro incubation with SM and pronase-catalyzed proteolysis, the alkylated amino acids Glu(-HETE) and His(-HETE) were detected. Both borne the SM-characteristic HETE-moiety bound to their side chain. The eightfold deuterated SM analog (d8-SM) was also applied to support adduct identification. Proteolysis conditions were optimized with respect to pH (8.0), temperature (50 C), and time to maximize the yield of Glu(-HETE) (30 min) and His(-HETE) (180 min). Amino acid adducts were stable in the autosampler for at least 24 h. Protein-adducts were stable in serum at À30 C for at least 33 days and for three freeze-and-thaw cycles. At the body temperature of chicken (+40 C), Glu(-HETE) was degraded in serum (period of half-change 3 days), whereas His(-HETE) remained stable. The presented method broadens the toolbox of procedures to document poisoning with SM.

Isolation of human TRPA1 channel from transfected HEK293 cells and identification of alkylation sites after sulfur mustard exposure

“…It is therefore plausible that alkylation at aspartic and glutamic acid in hTRPA1 had occurred. Both amino acids possess a free carboxylic group that can react with the electrophilic SM as already shown before (Smith et al 2008 ; John et al 2019 , 2022b ). Additionally, TRPC channels possess similar structures to hTRPA1 channels and have three to four ankyrin repeats at the N-terminus.…”

“…SM is known to alkylate thiol groups of cysteine residues by attaching a HETE -moiety (Lüling et al 2018 , 2021 ; John et al 2019 ; Schmeißer et al 2022 ) as well as carboxy-groups of aspartic acid and glutamic acid residues (John et al 2019 , 2022b ). Based on the peptide pattern detected from non-exposed hTRPA1, we calculated the masses of the protonated and potentially alkylated peptides for subsequent targeted μLC-ESI MS/HR MS (PRM) analysis of SM incubated samples.…”

“…John et al ( 2019 , 2022b ) showed that in addition to SM-induced modifications at cysteine residues, glutamic acid modifications were found in human and avian serum albumin. It is therefore plausible that alkylation at aspartic and glutamic acid in hTRPA1 had occurred.…”

Phosphonylated tyrosine and lysine residues as biomarkers of local exposure of human hair to the organophosphorus nerve agents sarin and VX

“…17,25 In contrast, peptide and amino acid adducts of SM and its analogues typically yield agentspecific diagnostic product ions in μLC-ESI MS/HR MS (PIS) analysis, for example, at m/z 105.0369. This ion represents the cleaved and charged hydroxyethyl thioethyl ([HETE] + ) moiety [30][31][32][33] making the detection of corresponding peptide adducts quite comfortable.…”