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.
| Michaelson-Richie, Erin D; Ming, Xun; Codreanu, Simona G et al. (2011) Mechlorethamine-induced DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. J Proteome Res 10:2785-96 |
| Michaelson-Richie, Erin D; Loeber, Rachel L; Codreanu, Simona G et al. (2010) DNA-protein cross-linking by 1,2,3,4-diepoxybutane. J Proteome Res 9:4356-67 |
| Loeber, Rachel L; Michaelson-Richie, Erin D; Codreanu, Simona G et al. (2009) Proteomic analysis of DNA-protein cross-linking by antitumor nitrogen mustards. Chem Res Toxicol 22:1151-62 |
| Liebler, Daniel C (2008) Protein damage by reactive electrophiles: targets and consequences. Chem Res Toxicol 21:117-28 |
| Lin, De; Saleh, Samir; Liebler, Daniel C (2008) Reversibility of covalent electrophile-protein adducts and chemical toxicity. Chem Res Toxicol 21:2361-9 |
| Lin, De; Saleh, Samir; Liebler, Daniel (2008) Reversibility of Covalent Electrophile-Protein Adducts and Chemical Toxicity. Chem Res Toxicol : |
| Wong, Hansen L; Liebler, Daniel C (2008) Mitochondrial protein targets of thiol-reactive electrophiles. Chem Res Toxicol 21:796-804 |
| Rachakonda, Girish; Xiong, Ying; Sekhar, Konjeti R et al. (2008) Covalent modification at Cys151 dissociates the electrophile sensor Keap1 from the ubiquitin ligase CUL3. Chem Res Toxicol 21:705-10 |
| Shin, Nah-Young; Liu, Qinfeng; Stamer, Sheryl L et al. (2007) Protein targets of reactive electrophiles in human liver microsomes. Chem Res Toxicol 20:859-67 |
| Orton, Christopher R; Liebler, Daniel C (2007) Analysis of protein adduction kinetics by quantitative mass spectrometry: competing adduction reactions of glutathione-S-transferase P1-1 with electrophiles. Chem Biol Interact 168:117-27 |
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