Identification and Significance of Protein Adducts

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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.