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 µg.mL⁻¹). Five new alkylation sites were accurately identified on the protein (α-His⁷², α-His⁸⁷, α-His⁸⁹, β-His² and β-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 α-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 µg.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 µg.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 β. 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.

硫芥子油是一种高反应性化学战剂，可通过烷基化多种生物分子（例如蛋白质）对受害的受害者造成严重损害。所得的烷基化产物可用作暴露于该化学试剂的生物标记。因此开发了液相色谱-串联质谱（LC-MS / MS）方法，以胰蛋白酶消化血红蛋白（50 mg.mL ^-1），并用不同浓度（0.25、0.5、1 ，10和100 µg.mL ^-1）。五个新的烷基化位点被精确地识别蛋白质上的（α-His的⁷²，α-His的⁸⁷，α-His的⁸⁹，β-His的²和β-VAL ⁹⁸）和15种加成肽，其中8种是由4种肽的烷基化产生的，每一种都具有两个潜在的加合位点，这些位点可以通过方法特异性加以区分。类似地，有可能区分肽α-T9的三个潜在的内含位点。此外，该方法允许以令人满意的重复性对所有烷基化的肽进行定量，当血红蛋白暴露于100 µg.mL ^-1的芥子气中时，RSD为0.5至9.3％。用不同浓度的芥菜芥子油温育的血红蛋白的分析导致所有烷基化肽的线性响应具有研究浓度（0.25、0.5、1、10和100 µg.mL ^-1）。在所研究的不同肽之间还观察到烷基化速率的变化，硫芥子在组氨酸残基上以及在球蛋白链的N端缬氨酸残基和球蛋白β的Val ⁹⁸残基上优先加成。此外，所提出的方法被证明是灵敏的，具有理论上的可能性来检测由去离子水中的血红蛋白与硫芥子在2.63 ng.mL ^-1下进行体外温育而产生的烷基化肽。经过进一步开发，该方法可能会用于分析暴露于硫芥末的体内血液样本。