Browsing by Subject "Carbonylation"

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    Improving the detection of carbonylated peptides by mass spectrometry via solid-phase hydrazide enrichment and selective labeling with Oxygen-18 (18O)
    (2010-01) Roe, Mikel Robert
    Protein carbonylation is a post-translational oxidative protein modification known to alter protein function and impair cellular mechanisms. It is a relatively complex modification, characterized by a variety of structurally distinct reactive carbonyls that target a number of amino acid residues and originate via several different oxidative mechanisms. While identification of specific carbonylated proteins by mass spectrometry has provided insight regarding the protein pathways and complexes affected, the specific sites of carbonyl modification, necessary for determining the oxidative mechanisms involved as well as for explaining any associated functional consequences, are not routinely identified due to the relatively low abundance of carbonylated proteins. To address this issue, a number of methods for enriching carbonylated peptides have been developed, all of which involve derivatization with bulky reagents that often complicate the identification of peptides by tandem mass spectrometry. As an alternative to these label-based approaches, I have developed a label-free method for enriching carbonylated peptides that is based on their selective capture and controlled release from a novel solid-phase hydrazide reagent (SPH). The value of the SPH reagent method is demonstrated using a yeast lysate treated with the reactive lipid carbonyl 4-hydroxynonenal (HNE), where the use of pulsed-Q-dissociation (PQD) and neutral-loss triggered MS/MS/MS was employed for the first time to assist the identification of HNE-modified peptides by mass spectrometry. To further improve the confidence by which carbonylated peptides are identified via mass spectrometry, a novel 18O-labeling method that selectively introduces an 18O molecule into the carbonyl oxygen of carbonylated peptides was developed. The resulting 18O isotope signature enables carbonylated peptide precursor ions and carbonylated MS/MS fragment ions to be tracked in the full-scan MS and MS/MS spectra, respectively, thus providing an independent validation of the MS/MS spectra matched to carbonylated peptides by proteomic database searching. The value of 18O-labeling for both improving the accuracy and measuring the efficiency of database-based identification of carbonylated peptides is demonstrated in an HNE-treated lysate from rat skeletal muscle homogenate. In conclusion, the development of the SPH reagent and the 18O-labeling method are useful tools for identifying carbonylated peptides in complex biological mixtures and represent important steps forward in the field of redox proteomics.
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    Protein carbonylation in the adipocyte nucleus
    (2017-11) Hauck, Amy
    As the incidence of obesity rises globally, it has become increasingly imperative to identify the mechanisms that cause obesity-related metabolic disease. In particular, oxidative stress in the adipose tissue is known to cause metabolic dysfunction, but the mechanisms that contribute to this process remain unclear. Protein carbonylation refers to the post-translational modification of lysine, cysteine, and histidine residues by diffusible electrophilic lipids. Specifically, 4- hydroxy-2-nonenal (4-HNE) and 4-hydroxy-2-hexenal (4-HHE) are produced at high levels in obese adipose tissue as a direct result of increased oxidative stress. The studies herein focus on the hypothesis that protein carbonylation is a mechanistic link between elevated oxidative stress and metabolic dysfunction in obese adipose tissue. We found that protein carbonylation is elevated specifically in the nucleus of adipocytes as a consequence of obesity and of aging. Proteomic evaluation of these modifications revealed that the core histones and zinc finger proteins are major targets of carbonylation. Since these proteins are critical regulators of transcriptional mechanisms, these data describe a potential link between oxidative stress and altered expression of metabolic pathways in adipose tissue.