Highly sensitive detection of S-nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence.

S-nitrosylated proteins are biomarkers of oxidative damage in aging and Alzheimer's disease (AD). Here, we report a new method for detecting and quantifying nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF). Dylight 488 maleimide was used to specifically label thiol group (SH) after switching the S-nitrosothiol (S-NO) to SH in cysteine using the "fluorescence switch" assay. In vitro nitrosylation model-BSA subjected to S-nitrosoglutathione (GSNO) optimized the labeling reactions and characterized the response of the LIF detector. The method proves to be highly sensitive, detecting 1.3 picomolar (pM) concentration of nitrosothiols in nanograms of proteins, which is the lowest limit of detection of nitrosothiols reported to date. We further demonstrated the direct application of this method in monitoring protein nitrosylation damage in MQ mediated human colon adenocarcinoma cells. The nitrosothiol amounts in MQ treated and untreated cells are 14.8±0.2 and 10.4±0.5 pmol/mg of proteins, respectively. We also depicted nitrosylated protein electrophoretic profiles of brain cerebrum of 5-month-old AD transgenic (Tg) mice model. In Tg mice brain, 15.5±0.4 pmol of nitrosothiols/mg of proteins was quantified while wild type contained 11.7±0.3 pmol/mg proteins. The methodology is validated to quantify low levels of S-nitrosylated protein in complex protein mixtures from both physiological and pathological conditions.

In-stent stenosis: potential role of increased oxidative stress and glutathione-linked detoxification mechanisms.

This study was designed to determine whether red-cell oxidative stress status and antioxidant enzyme levels can serve as markers in patients predisposed to in-stent stenosis. Blood was collected from patient groups undergoing coronary angiography for chest pain evaluation, namely, group A (without coronary artery disease), group B (previous coronary stents without in-stent stenosis), and group C (previous coronary stents with in-stent stenosis). Thiobarbituric acid reactive substances (measure of lipid peroxidation), glutathione-linked detoxification enzymes, catalase, and superoxide dismutase were determined. Compared with group A, patients in group C showed increased lipid peroxidation products and glutathione-S-transferase but decreased glutathione peroxidase and glutathione reductase activities. Results in group B patients were intermediate between those of groups A and C with significant decreases in glutathione peroxidase versus controls. In-stent stenosis is associated with significant increase in lipid peroxidation and attenuated glutathione-linked detoxification enzymes, consistent with oxidative stress.