Comprehensive Metabolomics by Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry: Integrative Strategies for Biomarker Discovery

Abstract

<p>Metabolomics is a rapidly emerging area of post-genomic research aimed at the comprehensive analysis of all low molecular weight metabolites in a cell or biofluid, which heralds new advances in drug development, disease prognosis and nutritional intervention. The major goals of this thesis are aimed at addressing several key obstacles hampering the development of metabolomics in biological research that is presented in four major chapters in this thesis. Chapter II introduces an integrative strategy for the identification of unknown low abundance metabolites when using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in conjunction with computer simulations when chemical standards are unavailable. The second project in Chapter III develops and validates an artifact-free method for analysis of labile metabolites in filtered red blood cell lysates by CE-ESI-MS that allows for accurate assessment of cellular redox status and antioxidant capacity based on the differential rates of glutathione oxidation. Chapter IV of the thesis introduces a differential metabolomics strategy for quantitative assessment of cellular oxidative stress and antioxidant efficacy with strenuous exercise. CE-ESI-MS together with univariate and multivariate data analysis was used to identify putative early- and late-stage biomarkers of oxidative stress in erythrocytes, which was applied to assess the attenuation of oxidative stress following highdose oral N-acetyl-L-cysteine administration. The final project in Chapter V investigates the impact of low-dose y-irradiation on intra-cellular metabolism and cell membrane viability of leukocytes when using CE-ESI-MS and fluorescencebased flow cytometry, respectively. A distinct non-linear cellular response was measured for irradiated-leukocytes as reflected by a significant upregulation of metabolites and slower progression of late-stage apoptosis when exposed to a minimum-threshold dose of radiation Metabolomics by CE-ESI-MS provides a novel and hypothesis-generating approach for investigating complex biological phenomena, which can improve our fundamental understanding of the underlying mechanisms of oxidative stress that is relevant to human health, aging and disease pathogenesis.</p>