Department of Analytical, Bioanalytical Sciences and Miniaturization (LSABM) Chemistry, Biology and Innovation (CBI), ESPCI Paris, PSL University, CNRS, 10 rue Vauquelin, 75005 Paris, France.
DGA, CBRN Defence, Analytical Chemistry Department, 5 rue Lavoisier, 91710 Vert-le-Petit, France.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jan 15;1163:122518. doi: 10.1016/j.jchromb.2020.122518. Epub 2020 Dec 25.
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) incubated with sulfur mustard at different concentrations (0.25, 0.5, 1, 10 and 100 µg.mL). Five new alkylation sites were accurately identified on the protein (α-His, α-His, α-His, β-His and β-Val) 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. 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). 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 Val 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. After further development, this method could potentially be used for the analysis of blood samples in vivo exposed to sulfur mustard.
芥子气是一种高反应性的化学战剂，可通过烷基化多种生物分子（如蛋白质）对暴露于其中的受害者造成严重损害。由此产生的烷基化产物可作为接触这种化学剂的暴露标志物。因此，开发了一种液相色谱串联质谱（LC-MS/MS）方法，用于检测经不同浓度（0.25、0.5、1、10 和 100 µg.mL）芥子气孵育后的血红蛋白（50 mg.mL）经胰蛋白酶消化后的烷基化肽。在蛋白质（α-His、α-His、α-His、β-His 和β-Val）上准确鉴定了五个新的烷基化位点，并检测到十五个加合物肽，其中八个是由四个肽的烷基化产生的，每个肽都有两个潜在的加合位点，该方法的特异性可将其区分开来。同样，可以区分肽α-T9 的三个潜在加合位点。此外，该方法可以定量所有烷基化肽，血红蛋白暴露于 100 µg.mL 芥子气时，重现性令人满意，RSD 为 0.5%至 9.3%。对不同浓度芥子气孵育的血红蛋白进行分析，导致所有研究浓度（0.25、0.5、1、10 和 100 µg.mL）下的所有烷基化肽均呈线性响应。还观察到研究的不同肽之间的烷基化率也存在差异，芥子气优先与组氨酸残基加合，但也与两条球蛋白链的 N-末端缬氨酸残基以及球蛋白β的 Val 残基加合。此外，所提出的方法具有较高的灵敏度，理论上有可能检测到在去离子水中与芥子气孵育的血红蛋白的体外孵育产生的烷基化肽，检测限为 2.63 ng.mL。进一步开发后，该方法有可能用于分析体内暴露于芥子气的血液样本。
