During the initial funding period of this PPG, we and our colleagues have implemented mass spectrometry(MS)-based proteomics technologies and novel affinity labeling and capture techniques to enable theselective capture and analysis of proteins labeled by specific lipid oxidation products. Our overall objectivein this competing renewal application is to define the scope of protein damage by lipid electrophiles in cellsand plasma and to apply sensitive MS and immunoaffinity methods to study protein adducts as modulatorsof biological responses to oxidative stress and as biomarkers for oxidative stress. A major thrust of theproposed work is an evaluation of the target selectivity and biological effects of lipid electrophiles in boththeir 'free' and phospholipid-esterified forms. These studies will collectively advance our understanding ofhow protein damage by lipid electrophiles transduces environmentally-induced oxidatve stress into discretesignaling effects and will identify new candidate biomarkers for oxidative damage in plasma and tissues.
The specific aims of Project 4 are: 1) To identify the protein targets of lipid electrophiles in cell models.These studies will characterize protein adduction by lipid electrophiles using Click chemistry-based methodsto capture and analyze protein and peptide adducts. 2) To identify potential damage-susceptible cellularsystems and candidate electrophile sensors by mapping electrophile-adducted proteins to cellularprocesses, canonical pathways, and protein-protein interaction networks archived in a local database. 3) Toidentify protein targets and corresponding adduction sites on proteins that regulate responses to oxidativestress in cells. This work will be done in collaboration with Project 3 to test the hypothesis that alkylation ofspecific proteins by lipid electrophiles triggers signaling and transcription factor regulation changesassociated with stress. 4) To evaluate apolipoprotein A1 (ApoA1) adducts as biomarkers of oxidative stress'n vivo. We will evaluate the hierarchical reactivity of ApoA1 nucleophiles with lipid electrophiles fromelectrophile probes and endogenous HDL lipids in plasma oxidations in vitro. Multiple reaction monitoring(MRM)-based LC-MS-MS quantitative assays will be developed for specific adducts, which will be evaluatedas biomarker candidates in vivo in human specimens in collaboration with Project 2.
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