Evidence of Sulfur Mustard Exposure in Human Plasma by LC-ESI-MS-MS Detection of the Albumin-Derived Alkylated HETE-CP Dipeptide and Chromatographic Investigation of Its Cis/Trans Isomerism

Abstract

Sulfur mustard (SM) is a chemical warfare agent that causes painful blisters and chemically modifies endogenous biomacromolecules by alkylation to hydroxyethylthioethyl (HETE) adducts representing valuable long-term markers for post-exposure analysis. The albumin adduct formed in human plasma in vitro (HETE bound to the side chain of cysteine 34) was isolated and cleaved by current lots of pronase primarily generating the internal modified dipeptide (HETE-cysteine-proline, HETE-CP) instead of the formerly reported HETE-CPF tripeptide. The analyte was detected by liquid chromatography-electrospray ionization tandem-mass spectrometry (LC-ESI-MS-MS). In principle, HETE-CP undergoes a dynamic on-column equilibrium of cis-trans isomerism thus requiring separation at 50°C to obtain one narrow peak. Accordingly, we developed both a novel longer lasting but more sensitive microbore (1 mm i.d., flow 30 µL/min, cycle time 60 min, LOD 50 nM) and a faster, less sensitive narrowbore (2.1 mm i.d., 200 µL/min, cycle time 16 min, LOD 100 nM, both on Atlantis T3 material at 50°C) LC-ESI-MS-MS method suitable for verification analysis. The corresponding tri- and tetrapeptide, Q(HETE)-CPF were monitored simultaneously. HETE-CP peak areas were directly proportional to SM concentrations added to plasma in vitro (0.05-100 µM). Albumin adducts formed by deuterated SM (d8-SM) served as internal standard.

Methionine329 in human serum albumin: A novel target for alkylation by sulfur mustard

“…These peptides documented their suitability as biomarkers in plenty of studies [16][17][18][19][20][21][22][23] and thus represent highly valuable markers for SM exposure. After proteolysis with pronase and ProtK the alkylated peptides HETE-CP and HETE-CPF are generated, respectively.…”

“…Therefore, toxicity of SM is assumed to be due to the alkylation of proteins and DNA resulting in the functional impairment of cells, tissues, organs, and physiological processes. 12,[17][18][19][20][21][22][23] Based on the proteolysis of alkylated HSA, several analytical methods targeting the Cys 34 modification as HETE-CysProPhe (HETE-CPF) and HETE-CysPro (HETE-CP) were developed and have proven to be reliable biomarkers of exposure in vitro, in vivo, and in real cases of SM poisoning. 2 For the verification of an alleged use of SM, protein adducts reveal beneficial long-term stability of several weeks or even months in vivo when compared to products of SM hydrolysis and enzymatic biotransformation.…”

“…Due to its bifunctional structure, crosslinks between biomolecules may occur. 12,[15][16][17][18][19][20][21][22][23][24] Herein, we present Met 329 as an additional target of SM in HSA. 15,16 Most relevant biomarkers are alkylated peptides derived from human serum albumin (HSA).…”

Modification of human serum albumin by the nerve agent VX: microbore liquid chromatography/electrospray ionization high‐resolution time‐of‐flight tandem mass spectrometry method for detection of phosphonylated tyrosine and novel cysteine containing disulfide adducts

“…It was shown that certain tyrosine residues are prone to phosphylation (denominating both phosphorylation and phosphonylation) by numerous OP and that Cys 34 – the only amino acid containing a free thiol group – can easily be modified by electrophiles (e.g. by alkylation with sulfur mustard) . Subsequent enzymatic cleavage of HSA allows generation of small cleavage products, which are analyzed using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS).…”

Identification of novel disulfide adducts between the thiol containing leaving group of the nerve agent VX and cysteine containing tripeptides derived from human serum albumin

“…While phosphylation is the critical step of AChE inhibition by modification of its active site serine residue, we have recently shown that the corresponding leaving group of VX (2-(diisopropylamino)ethanethiol, DPAET) is also capable of forming adducts with endogenous cysteine residues of HSA [25] . Cys-Pro dipeptides including the only free cysteine sidechain at position 34 (C 34 P) were already known to result from pronase cleavage of HSA and facilitated the identification of DPAET-CP as a novel biomarker for VX exposure in vitro [8,[14][15][16][17]25] . Enzymatic cleavage of HSA incubated with VX using pronase resulted in cysteine and proline containing small peptides.…”