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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES010056-04
Application #
6525216
Study Section
Special Emphasis Panel (ZRG1-CPA (03))
Program Officer
Balshaw, David M
Project Start
1999-09-15
Project End
2003-12-01
Budget Start
2002-09-01
Budget End
2003-12-01
Support Year
4
Fiscal Year
2002
Total Cost
$213,491
Indirect Cost
Name
University of Arizona
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721
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
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Liebler, Daniel C (2008) Protein damage by reactive electrophiles: targets and consequences. Chem Res Toxicol 21:117-28
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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