Protein targets and metabolites of 1,1-dichloroethylene

Abstract

Xenobiotics and their metabolites are capable of covalent modification of proteins that may lead to cellular injury. Determining the identity of modified proteins, however, has to date been hindered by available analytical instrumentation. Advances in mass spectrometry now allow for routine analysis of large biomolecules. This has given rise to the field of proteomics , which uses mass spectrometry in conjunction with various separation techniques to gain information about large numbers of proteins. This dissertation describes the development of a new proteomic methodology to identify unknown protein targets and its application to identifying protein targets and metabolites of 1,1-dichloroethylene (1,1-DCE). 1,1-DCE undergoes P450 bioactivation in hepatocytes to produce 1,1-DCE oxide and 2-chloroacetyl chloride. These either modify proteins directly, or form the glutathione conjugate S-(2-chloroacetyl)glutathione, which in turn is capable of protein cysteinyl sulfhydryl alkylation. In animals, exposure to 1,1-DCE results in selective injury to the biliary canalicular membrane. Biochemical and physiological evidence suggests damage to the canalicular membrane transport proteins. Putative 1,1-DCE metabolite adducts, including S-carboxymethylated and 2-chloroacetylated peptides and GSCOCH₂-S-cys-peptide adducts, were synthesized using model peptides containing one or two cysteines. The adducts were analyzed by ESI-MS-MS and resulted in fragmentation patterns characteristic of the adduct moiety, including fragment ions, losses from the parent ion and pairs of ions separated by the mass of the modified cysteine residue. The data reduction algorithm SALSA was developed to search for user-specified fragmentation characteristics in MS-MS data dependent scans. Bile samples obtained from animals exposed to 1,1-DCE were analyzed by ESI-MS-MS. SALSA was then used to search the data for spectra containing adduct-specific fragmentation. Five hepatic S-carboxymethylated proteins were identified, as were the 1,1-DCE metabolites S-carboxymethylglutathione, S-(cysteinylacetyl)glutathione and the cyclic product of the intermolecular rearrangement of S-(2-chloroacetyl)glutathione. This work demonstrates the use of mass spectrometry to characterize unknown, modified proteins in complex mixtures without the use of radio- or immunochemical labeling.