Date of Award

Fall 2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering

First Advisor

June Feng

Abstract

Protein S-nitrosylation, the covalent modification of a cysteine sulfhydryl group by nitric oxide (NO), plays a critical role in post-translational modification (PTM) that regulates a large variety of cellular functions and signalling events. The nitrosylation state changes with oxidative damage and is involved in variety of cancers and neurodegeneration diseases.

Three technologies were developed for nitrosylated protein detection and identification. Capillary gel electrophoresis with laser induced fluorescence (CGE-LIF) detection was used for the detection and quantitation of nitrosylated proteins. A two-dimensional micro-electrophoresis (2D μ-CE) separations system was also built to detect nitrosylated proteins via poly (methylmethacrylate) microchips. Proteomics following nitrosylated proteins enrichment was used for identification of protein S-nitrosylation.

The CGE-LIF method, with a limit detection of 1.3 picomolar (pM) concentration of nitrosothiols in nanograms of proteins, is the lowest limit of detection of nitrosothiols reported to date. 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 direct application of this method was demonstrated in monitoring protein nitrosylation damage in menadione (MQ) mediated human colon adenocarcinoma cells (HT-29). The nitrosothiol amounts in 200 μM MQ treated and untreated cells are 14.8±0.2 and 10.4±0.5 pmol/mg of proteins, respectively. In addition, also depicted are nitrosylated protein electrophoretic profiles of brain cerebrum of five-month-old AD transgenic (Tg) mice model. In the Tg mice brain, 15.5±0.4 pmol of nitrosothiols/mg of proteins was quantified while the 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.

Compared with the conventional protein separation method, microfluidics has been widely used for development of novel tools for separation of protein mixtures. Sodium dodecyl sulphate micro-capillary gel electrophoresis (SDS μ-CGE) and microemulsion electrokinetic chromatography (MEEKC) were used for the 1D and 2D separations, respectively. The effective separation lengths for both dimensions were ten mm, and electrokinetic injection was used with field strength at 200V/cm. After 80 seconds separation in the 1D CGE, fractions were successfully transferred to a second MEEKC dimension for a short ten-seconds separation. This 2D μ-CE separation was first demonstrated by resolving five standard proteins with a molecular weight (MW) ranging from 20 to 64 kDa. A high peak capacity 3D landscape image of nitrosylated proteins from HT-29 cells before and following MQ treatment to induce oxidative stress is also presented. Additionally, to illustrate the potential of the 2D μ-CE separation method for rapid profiling of oxidative stress-induced biomarkers implicated in AD disease, the nitrosylated protein fingerprints from 11-month-old AD transgenic mice brain and its age matched control were also generated. This is the first known report on 2D profiling of nitrosylated proteins in the biological samples on the microchip. The characteristics of this biomarker profiling will potentially serve as the screening for early detection of AD.

The proteomics study revealed protein S-nitrosylation in the MQ treated human colon adenocarcinoma, HT-29 cells. Its profile of S-nitrosylated proteins was compared to a control cell line not exposed to MQ. A total of 20 proteins were modified by nitrosylation in MQ-treated HT-29 cells and at least ten nitrosylated proteins were increased by exposure to MQ. Seventeen of the 20 proteins are novel targets of S-nitrosylation not previously reported. These proteins include cytoskeletal and signaling proteins, metabolic enzymes, chaperones, and redox-and differentiation-related proteins. These results broaden knowledge of therapy targets.

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