New research and publication on the adduct of the blistering agent Sesquimustard with Human Serum Albumin

As the result of a scientific collaboration with the Bundeswehr Institut of Pharmacology and Toxicology we have jointly published a research article in the journal Analytical and Bioanalytical Chemistry, which was selected by the editors to become a “Paper in Forefront” (according to the journal Papers in Forefront are exceptional papers that the Editors selected guided by peer review).

In this paper we report on the adduct of the blistering agent Sesquimustard with Human Serum Albumin (HSA) and its mass spectrometric identification for biomedical verification of exposure. Why is this important?

In addition to the well known chemical warfare agent sulfur mustard (SM) a range of higher sulfur mustards exist that are also listed in Schedule 1 of the Chemical Weapons Convention and show stronger blistering properties than SM. Sesquimustard is regularly found as a common impurity in mustard mixtures and in old munitions but can also be used in pure form.

Victims of an exposure to Sesquimustard would show the same symptoms as those exposed to regular sulfur mustard but a bioanalytical investigation would come back negative as the typical biomarkers (including protein biomarkers) of sulfur mustard would be missing.

We 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.

The paper is available open-access on the journal’s homepage.

Adduct of the blistering warfare agent sesquimustard with human serum albumin and its mass spectrometric identification for biomedical verification of exposure
Marc-Michael Blum, Annika Richter, Markus Siegert, Horst Thiermann & Harald John
Analytical and Bioanalytical Chemistry (2020), 412:7723-7737.