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 the selective capture and analysis of proteins labeled by specific lipid oxidation products. Our overall objective in this competing renewal application is to define the scope of protein damage by lipid electrophiles in cells and plasma and to apply sensitive MS and immunoaffinity methods to study protein adducts as modulators of biological responses to oxidative stress and as biomarkers for oxidative stress. A major thrust of the proposed work is an evaluation of the target selectivity and biological effects of lipid electrophiles in both their free and phospholipid-esterified forms. These studies will collectively advance our understanding of how protein damage by lipid electrophiles transduces environmentally-induced oxidatve stress into discrete signaling 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 methods to capture and analyze protein and peptide adducts. 2) To identify potential damage-susceptible cellular systems and candidate electrophile sensors by mapping electrophile-adducted proteins to cellular processes, canonical pathways, and protein-protein interaction networks archived in a local database. 3) To identify protein targets and corresponding adduction sites on proteins that regulate responses to oxidative stress in cells. This work will be done in collaboration with Project 3 to test the hypothesis that alkylation of specific proteins by lipid electrophiles triggers signaling and transcription factor regulation changes associated 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 from electrophile 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 evaluated as biomarker candidates in vivo in human specimens in collaboration with Project 2.