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 34 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 Cys 34 *Pro and the single amino acid residue His* were produced. The use of proteinase K yielded Cys 34 *ProPhe and the use of pepsin generated ValThrGlu 48 *Phe, AlaGlu 230 *ValSerLysLeu, and LeuGlyMet 329 *Phe. 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 230 and Met 239 residues were detected and are reported accordingly. Based on molecular dynamics simulations, we identified regular interactions of the Cys 34 (-HETEOETE)-moiety with several glutamic acid residues in HSA including Glu 86 , 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.