The blistering warfare agent O-mustard (agent T) generates protein-adducts with human serum albumin useful for biomedical verification of exposure and forms intramolecular cross-links.

The highly blistering sulfur mustard analogue agent T (bis(2-chloroethylthioethyl) ether), also known as O-mustard or oxy-mustard, is a common impurity in military grade sulfur mustard (SM) and a component of mixtures such as "HT" that are still found in old munitions. Together with sesquimustard (Q), it is the most important SM analogue and tightly regulated as a Schedule 1 chemical under the Chemical Weapons Convention. We report the adducts of T with nucleophilic Cys and other residues in human serum albumin (HSA) formed in vitro. A micro liquid chromatography electrospray ionization high-resolution tandem-mass spectrometry method (µLC-ESI MS/HR MS) was developed for the detection and identification of biomarker peptides alkylated by a T-derived hydroxyethylthioethyloxyethylthioethyl (HETEOETE)-moiety (as indicated by an asterisk below). Following proteolysis of T-exposed human plasma with pronase, the dipeptide CysPro and the single amino acid residue His were produced. The use of proteinase K yielded CysProPhe and the use of pepsin generated ValThrGluPhe, AlaGluValSerLysLeu, and LeuGlyMetPhe. Corresponding peptide-adducts of SM and Q were detected in a common workflow that in principle allowed the estimation of the mustard or mustard composition encountered during exposure. Novel adducts of Q at the Glu and Met residues were detected and are reported accordingly. Based on molecular dynamics simulations, we identified regular interactions of the Cys(-HETEOETE)-moiety with several glutamic acid residues in HSA including Glu, which is not an obvious interaction partner by visual inspection of the HSA crystal structure. The existence of this and other intramolecular cross-links was experimentally proven for the first time.