Mass spectrometry-based identification and characterization of protein and peptide adducts of lipoxidation-derived aldehydes Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/r781wh96m

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  • Oxidative stress is recognized as an important underlying factor in the pathogenesis of many degenerative diseases as well as normal senescence. The free radicals, reactive oxygen species (ROS) and electrophiles produced during oxidative stress are capable of modifying nucleic acids, lipids and proteins. There are a variety of oxidative modifications that occur to proteins including: cleavage of the protein backbone, direct oxidation of amino acid side chains by ROS, and adduction by electrophilic species such as lipid peroxidation products. Many of these oxidative modifications result in the introduction of carbonyl groups into the proteins. Protein carbonylation levels are commonly used as a biomarker to assess the degree of oxidative damage to a system. However the most commonly employed methods for measuring oxidative modifications to proteins, typically fail to provide any information about the identity of the modified protein, site of modification, or the chemical nature of the modification. In the present study we develop an analytical technique based on affinity labeling with N'-aminooxymethylcarbonylhydrazino-D-biotin (aldehyde reactive probe, ARP), along with mass spectrometric analysis which allows for the full characterization of protein carbonylation modifications. The ability of the ARP method was first demonstrated for the case of oxylipid peptide and protein conjugates formed by Michael addition-type conjugation reactions with α,β- unsaturated aldehydic lipid peroxidation products with nucleophilic amino acid residue side chains. ARP was used to label a 4-hydroxy-2-nonenal (HNE) modified cysteine containing model peptide, and HNE modified E. coli thioredoxin, which were characterized using ESI-MS/MS and MALDI-MS/MS. ARP was also used to label the oxidative modifications alpha-aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS), formed during the metal catalyzed oxidation of GAPDH. After demonstrating the utility of the technique on model systems, it was then applied to complex biological systems. In one case, subsarcolemmal mitochondria (SSM) isolated from rat cardiac tissue. Mitochondria are well known to be a major source of ROS within the cell. They are therefore important mediators of oxidative stress, as well as regulators of cell death. We were able to identify 39 unique sites on 27 mitochondrial proteins which were modified by six different α,β-unsaturated aldehydes, including acrolein, β-hydroxyacrolein, crotonaldehyde, 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal and 4-oxo-2- nonenal. Additionally we identified nine Lys residues on four mitochondrial proteins that were oxidized to AAS and subsequently labeled with ARP. The proteins identified with oxidative modifications include members of the mitochondrial electron transport chain, TCA cycle, membrane transport, lipid metabolism, and other important mitochondrial enzymes. The ARP technique was also applied to identify protein targets of 4-hyroxy-2- nonenal in human monocytic THP-1 cells that were exogenously exposed to HNE. It was shown previously that exposure of THP-1 cells to HNE resulted in apoptosis, necrosis and protein carbonylation. We applied a multi-pronged proteomic approach involving electrophoretic, immunoblotting and mass spectrometric analysis to unequivocally identify eighteen sites of HNE modification on sixteen proteins. It was also demonstrated in this study that pretreatment of THP-1 cells with ascorbic acid resulted in decreased levels of HNE-protein conjugate formation.
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