Lipid hydroperoxide-derived bifunctional electrophiles, such as 4-HNE, arise in cells under conditions of oxidative stress, can covalently modify proteins and have been associated with a number of important pathological conditions. Identification of these protein targets will shed light on the origins and mechanisms of these pathologies. Current proteomic techniques are limited by their inability to analyze post-translational protein modifications. This proposal, which has three Specific Aims, offers a gel-free proteomic approach to identify lipid modified proteins in mitochondria. The methodology would rely on subcellular fractionation and SPE-based amplification of digested modified proteins to generate a peptide library, enriched with modified peptides. LC/MS analysis will be used to analyze peptide fragments and identify modified proteins. The methodology will be developed using a selected protein of interest that undergoes modification by bifunctional electrophiles. Once optimized, this approach will be used to identify proteins from isolated mitochondria, treated in vitro with synthetic bifunctional electrophiles. Next, mitochondria from A549 cells, expressing 15-lipoxygenase, grown under normal or oxidative stress conditions, will be analyzed for the presence of lipid-modified proteins. Identification of modified proteins that are also involved in apoptosis, resulting from oxidative stress, will demonstrate the involvement of bifunctional electrophiles in cell death and offer new insights in the role of lipid peroxidation in cell pathophysiology.
