Proteins have long been appreciated as critical targets of environmental chemicals that produce adverse health effects. Recent developments in mass spectrometry (MS) ionization methods and instrumentation now make possible the rapid, high throughput analysis of proteins. The goal of this application is to develop and refine current state-of-the-art technology for the global detection of low abundance protein post-translational modifications (PTMs) induced by environmental chemicals. We hypothesize that topological, chemical and physical features combine to determine which proteins are targets for chemical adduction, and such adduction causes a change in structure/function of the protein which subsequently contributes to the toxicological response to chemical exposure. These PTMs are present at low abundance and are not detectable by conventional protocols. MS approaches to specifically analyze low abundance PTMs are currently being developed in our laboratory. A series of isolation and enrichment steps combined with selective detection with MS and spectral processing algorithms will be used for global identification of PTMs. Adducted proteins will be isolated by immune-precipitation, separated by 2D gel electrophoresis and analyzed by MALDI and multidimensional I-IPLC-ESI-MS/MS. SALSA (Scoring ALgorithm for Spectral Analysis) will be used to characterize sites of protein modification. With well defined sites of adduction we will then determine the consequences of protein adduction. The goals of this application are to (i) develop MS methods to identify protein targets of environmental chemicals, (ii) ascertain those features that predispose certain proteins, or motifs within proteins (""""""""electrophile binding motifs"""""""") to chemical adduction, and (iii) to determine the potential biological/toxicological consequences of adduction to rationally selected """"""""electrophilins"""""""". For the latter aim, we will utilize a well characterized model of chemical-induced toxicity to determine the specific effects of chemical-induced PTMs on (a) the function of nuclear actin and the effects of PTM-actin on chromatin remodeling, and (b) the function of cytochrome c, and the effects of PTM-cytochrome c on the initiation and progression of apoptosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070890-03
Application #
7228818
Study Section
Special Emphasis Panel (ZRG1-BECM (01))
Program Officer
Okita, Richard T
Project Start
2005-03-01
Project End
2009-02-28
Budget Start
2007-03-01
Budget End
2008-02-29
Support Year
3
Fiscal Year
2007
Total Cost
$258,456
Indirect Cost
Name
University of Arizona
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
806345617
City
Tucson
State
AZ
Country
United States
Zip Code
85721
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Fisher, Ashley A; Labenski, Matthew T; Monks, Terrence J et al. (2011) Utilization of MALDI-TOF to determine chemical-protein adduct formation in vitro. Methods Mol Biol 691:303-16
Lau, Serrine S; Kuhlman, Christopher L; Bratton, Shawn B et al. (2010) Role of hydroquinone-thiol conjugates in benzene-mediated toxicity. Chem Biol Interact 184:212-7
Leinweber, Barbara D; Tsaprailis, George; Monks, Terrence J et al. (2009) Improved MALDI-TOF imaging yields increased protein signals at high molecular mass. J Am Soc Mass Spectrom 20:89-95

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