Differential Carbonylation of Proteins as a Function of in vivo Oxidative Stress

Abstract

This study reports for the first time qualitative and quantitative differences in carbonylated proteins shed into blood as a function of increasing levels of OS. Carbonylated proteins in freshly drawn blood from pairs of diabetic and lean rats were derivatized with biotin hydrazide, dialyzed, and enriched with avidin affinity chromatography. Proteins thus selected were used in several ways. Differences between control and diabetic subjects in relative concentration of proteins was achieved by differential labeling of tryptic digests with iTRAQ™ reagents followed by reversed phase chromatography (RPC) and tandem mass spectrometry (MS/MS). Identification and characterization of OS induced post-translational modification sites in contrast was achieved by fractionation of affinity selected proteins before proteolysis and RPC-MS/MS. Relative quantification of peptides bearing oxidative modifications was achieved for the first time by selective reaction monitoring (SRM). Approximately 1.7% of the proteins in Zucker diabetic rat plasma were selected by the avidin affinity column as compared to 0.98% in lean animal plasma. Among the thirty five proteins identified and quantified, Apo AII, clusterin, hemopexin precursor and potassium voltage-gated channel subfamily H member 7 showed the most dramatic changes in concentration. Seventeen carbonylation sites were identified and quantified, eleven of which changed more than 2 fold in oxidation state. Three types of carbonylation were identified at these sites; direct oxidative cleavage from reactive oxygen species, glycation and addition of advanced glycation end products, and addition of lipid peroxidation products. Direct oxidation was the dominant form of carbonylation observed while hemoglobin and murinoglobulin 1 homolog were the most heavily oxidized proteins.

Influence of Ascorbic Acid Supplementation on Type 2 Diabetes Mellitus in Observational and Randomized Controlled Trials; A Systematic Review with Meta-Analysis

“…Under diabetic conditions, ROS are produced mainly through the glycation reaction. Oxidative stress can in turn promote glycation of hemoglobin (6) and impair the ability of β-cells of the pancreas for insulin secretion (7). _________________________________________ On the other hand, several epidemiological studies have shown that individuals with low concentration of antioxidants are at increased risk of diabetes complications (8,9).…”

The alteration in peripheral neutrophils of patients with chronic kidney disease

“…As individual proteins have different amount of carbonylation sites [10], the diminution of proteins with low capability to react with dinitrophenylhydrazine can be connected with the previous formation of oxidized proteins.…”

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.…”