Determining the Effects of Antioxidants on Oxidative Stress Induced Carbonylation of Proteins

Abstract

There is potential that the pathological effects of oxidative stress (OS) associated diseases such as diabetes could be ameliorated with antioxidants, but this will require a clearer understanding of the pathway(s) by which proteins are damaged by OS. This study reports the development and use of methods that assess the efficacy of dietary antioxidant supplementation at a mechanistic level. Data reported here evaluate the impact of green tea supplementation on oxidative stress induced post-translational modifications (OSi-PTMs) in plasma proteins of Zucker diabetic fatty (ZDF) rats. The mechanism of antioxidant protection was examined through both the type and amount of OSi~PTMs using mass spectrometry based identification and quantification. Carbonylated proteins in freshly drawn blood samples were derivatized with biotin hydrazide. Proteins thus biotinylated were selected from plasma samples of green tea fed diabetic rats and control animals by avidin affinity chromatography, further fractionated by reversed phase chromatography (RPC), fractions from the RPC column were tryptic digested, and the tryptic digest fractionated by RPC before identified by tandem mass spectrometry (MS/MS). Relative quantification of peptides bearing carbonylation sites was achieved for the first time by RPC-MS/MS using selective reaction monitoring (SRM). Seventeen carbonylated peptides were detected and quantified in both control and treated plasma. The relative concentration of eight was dramatically different between control and green tea treated animals. Seven of the OSi-PTM bearing peptides had dropped dramatically in concentration with treatment while one increased, indicating differential regulation of carbonylation by antioxidants. Green tea antioxidants were found to reduce carbonylation of proteins by lipid peroxidation end products most, followed by advanced glycation end products to a slightly lower extent. Direct oxidation of proteins by reactive oxygen species (ROS) was protected the least by green tea.

Post-translational Modifications and Mass Spectrometry Detection

“…During the time period under study we could only find the works published by Regnier and co-workers describing sample enrichment in carbonylated proteins. Selection of carbonylated proteins was achieved through the derivatization of the carbonyl moiety with biotin hydrazine followed by avidin affinity chromatography (220,264). Despite these recent efforts, in the articles herein compiled, the number of oxidative modifications and oxidized proteins identified is generally small when compared to the numbers available for other modifications such as phosphorylation or acetylation.…”

Examination of the Efficacy of Antioxidant Food Supplements Using Advanced Proteomics Methods

“…Recent studies have shown that it is possible to study the efficacy of antioxidant supplementation at the level of oxidation sites using selective reaction monitoring (SRM) techniques (Madian et al, 2011b). As a proof of concept, the efficacy of green tea on OSi-PTMs in plasma proteins of Zucker diabetic fatty (ZDF) rats was evaluated (Figs.…”

“…As seen, the levels were reduced 25-fold in the pooled plasma sample from green tea-fed diabetic rats compared to the pooled plasma sample from control diabetic animals. Reprinted with permission from Madian et al (2011b). Copyright 2011 American Chemical Society.…”

“…Each sequence was uploaded into the Modeling Workspace where the software finds the best structure template based on homology with other proteins of known structure. Reprinted with permission from Madian et al (2011b). Copyright 2011 American Chemical Society.…”

Oxidative Stress in Human Health and Disease

“…The  cell is insulin independent for glucose uptake so under elevated plasma glucose, the cells fail to down regulate glucose entry by insulin resistance. Free available reducing sugars (eg.glucose), can react with free amino groups to form a Schiffs base which rearranges into an Amadori glycation product (158)(159)(160). When accumulated in proteins, these AGEs modify protein function and or contribute to generation of ROS thereby damaging the cell (151).…”