Proteins long have been appreciated as critical targets of environmental chemicals that produce toxicity and cancer. Recent developments in mass spectrometry ionization methods and instrumentation have driven the growth of proteomics, the study of the protein complement of the genome. The overall goal of this project is to use mass spectrometry methods for proteomics to identify the protein targets of environmental chemicals. We propose to selectively identify adducted peptides derived from proteolysis of adducted proteins and to characterize adduction at the level of amino acid sequence. We will use these approaches to test the hypothesis that specific cellular patterns of toxicity are linked to covalent modification of specific protein targets.
In specific aim 1, we will develop methods for detecting and identifying protein targets of environmental chemicals. Work in this aim is sub-divided into four major emphasis areas. la) Peptide adduct libraries will be prepared from synthetic peptides site-specifically adducted by the reactive intermediates 1,4-benzoquinone, dehydromonocrotaline, 4-vinylcyclohexene diepoxide, and reactive glutathione conjugates derived from 1,1-dichloroethylene and used to establish characteristic MS-MS fragmentation of adducted peptides. lb) Instrument control algorithms will be developed for rapid, selective detection and sequencing of adducted peptides in HPLC-MS-MS analyses. 1c) Proteomics analysis methods based on electrophoresis- or HPLC-based separations, combined with proteolytic digestion and MS-MS analysis will be adapted to the identification of adducts. 1d) The applications of """"""""shotgun sequencing"""""""" methods to the identification of protein adducts in complex mixtures by electrospray HPLC-MS-MS will be explored.
In specific aim 2, we will characterize the protein targets of 1,1-dichloroethylene (DCE) in rat liver. We will identify DCE-derived adducts with hepatocyte canalicular membrane to test the hypothesis that specific adduction leads to selective canalicular injury. The methods developed in this project will open important, yet largely unexplored aspects of chemical biology to mechanistic investigation.
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