Disulfide-adducts with cysteine residues in human serum albumin prove exposure to malodorous mercaptans in vitro

Malodorants are mixtures containing mercaptans, which trigger the flight instinct upon exposure and might thus be deployed in military and civilian defense scenarios. Exposure to mercaptans might lead to unconsciousness, thus representing a possible threat for health. Therefore, we developed and validated a bioanalytical procedure for the simultaneous detection and identification of corresponding biomarkers for the verification of exposure to mercaptans. Disulfide-adducts of ethyl mercaptan (SEt), n-butyl mercaptan (SⁿBu), tert-butyl mercaptan (S^tBu) and iso-amyl mercaptan (SⁱAm) with cysteine (Cys) residues in human serum albumin (HSA) were formed by in vitro incubation of human plasma. After pronase-catalyzed proteolysis, reaction products were identified as adducts of the single amino acid Cys and the dipeptide cysteine-proline (Cys³⁴Pro) detected by a sensitive μLC-ESI MS/MS method working in the scheduled multiple reaction monitoring (sMRM) mode. Dose-response studies showed linearity for the yield of Cys³⁴Pro-adducts in the range from 6 nM to 300 μM of mercaptans in plasma and limits of identification (LOI) were in the range from 60 nM to 6 μM. Cys³⁴-adducts showed stability for at least 6 days in plasma (37 °C). The presented disulfide-biomarkers expand the spectrum for bioanalytical verification procedures and might be helpful to prove exposure to malodorants.