Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of tryptic digest of human hemoglobin exposed to sulfur mustard

Sulfur mustard is a highly reactive chemical warfare agent that causes severe damages to the victims exposed by alkylating multiple biomolecules such as proteins. Resulting alkylated products can be used as biomarkers of exposure to this chemical agent. A liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was thus developed to detect alkylated peptides after the tryptic digestion of hemoglobin (50 mg.mL(-1)) incubated with sulfur mustard at different concentrations (0.25, 0.5, 1, 10 and 100 mu g.mL(-1)). Five new alkylation sites were accurately identified on the protein (alpha-His72, alpha-His87, alpha-His89, beta-His2 and beta-Val98) and fifteen adducted peptides were detected, among which eight of them resulted from the alkylation of four peptides, each presenting two potential sites of adduction that could be discriminated by the method specificity. Similarly, it was possible to discriminate the three potential adduction sites of the peptide alpha-T9. Moreover, the method allowed the quantification of all the alkylated peptides with a satisfying repeatability, with RSD ranging from 0.5 to 9.3% for an exposure of hemoglobin to sulfur mustard at 100 mu g.mL(-1). The analysis of hemoglobin incubated with different concentrations of sulfur mustard levels led to a linear response for all the alkylated peptides with the studied concentrations (0.25, 0.5, 1, 10 and 100 mu g.mL(-1)). A variation of the alkylation rate was also observed between the different peptides studied, with a preferential adduction of sulfur mustard on the histidine residues but also on the N-terminal valine residues of both globin chains and on the Val98 residue of globin beta. Furthermore, the presented method proved to be sensitive, with a theoretical possibility to detect alkylated peptides resulting from in vitro incubation of hemoglobin in deionized water with sulfur mustard at 2.63 ng.mL(-1). After further development, this method could potentially be used for the analysis of blood samples in vivo exposed to sulfur mustard.

Automated summary

A new LC-MS/MS method was developed to detect alkylated peptides resulting from exposure to sulfur mustard. The method accurately identified alkylated sites on proteins and allowed for quantification with satisfying repeatability. It also showed sensitivity towards detecting alkylated peptides resulting from in vitro incubation, suggesting its potential for future analysis of blood samples exposed to sulfur mustard in vivo.