Tandem MS analysis of model peptide adducts from reactive metabolites of the hepatotoxin 1,1-dichloroethylene

Dichloroethylene (DCE) is a hepatotoxin that undergoes cytochrome P450-catalyzed bioactivation in hepatocytes to form 2-chloroacetyl chloride and I,l-dichloroethylene oxide. 8-Chloroacetyl chloride reacts with nucleophilic residues and with N-terminal amines to produce 2-chloroacetylated residues and with glutathione to form the reactive electrophile S-(2-chloroacetyl)glutathione (ClCH(2)COSG), which, in turn, is capable of sulfhydryl alkylation. 1,1-DCE oxide can bind to cysteine sulfhydryl groups and subsequently hydrolyze to form an S-carboxymethylated cysteine residue. S-Carboxymethylated, 2-chloroacetylated, and GSCOCH(2-)S-Cys-peptide adducts of model cysteine-containing peptides were synthesized, and their fragmentation patterns were characterized by electrospray tandem mass spectrometry. Synthesis of GSCOCH(2)-S-Cys-peptide adducts was achieved via a novel tert-butoxycarbonyl (tBOC) derivative of ClCH(2)COSG. CID of GSCOCH(2)-S-Cys-peptide adducts resulted in product ions and neutral losses indicative of the GSCOCH(2)-S-Cys moiety as well fragment ion pairs in the b- and y-ion series corresponding to the modified cysteine residue. S-Carboxymethylated peptides exhibited only a characteristic b- or y-series ion pair separated by 161 Da, corresponding to cysteine + CH2COOH. CID of 2-chloroacetylated peptides showed neutral losses of 36 (HCl), 78 (HCOCH2Cl), 96 (HCOCH2Cl + H2O), and 114 Da (HCOCH2Cl + 2H(2)O). Combinations of characteristic fragment, ions, neutral losses, and ion pairs thus are characteristic for DCE-derived adducts. These features can be used in an MS/MS data reduction algorithm for the selective identification of protein targets of DCE metabolites.