Applications of Capillary Electrophoresis and Mass Spectrometry for Bioanalytical Chemistry

Abstract

This thesis explores the capabilities of capillary electrophoresis (CE) in analyzing various biomolecules for analytical applications. The first research chapter demonstrates the detection, separation and quantification of cannabinoids (small molecules) using Capillary Zone Electrophoresis coupled with UV detection. 14 cannabinoid standards were separated and their migration times and absorption spectra were determined. This was then applied to analyze cannabinoid content in herbal cannabis extracts, detecting 14 cannabinoids in one short run. The second research chapter presents the hyphenation of CE to mass spectrometry (CE-MS) for cationic metabolome analysis of exosomes derived from breast cancer cells. Exosomes are extracellular vesicles released by cells that contain proteins, lipids, nucleic acids and metabolites indicative of their cell of origin. CE-MS enabled the separation and identification of over 200 cationic metabolites in exosome samples. Variables were observed in carnitine synthesis and purine metabolism pathways between cancerous and non-cancerous exosomes. This demonstrated the potential of CE-MS for biomarker discovery directly from exosomes. The third research chapter highlights CE applications in analyzing large macromolecules. Specifically, CE was used for the selection of DNA aptamers against the nucleoprotein of SARS CoV 2, the virus causing the COVID-19 pandemic. Two SARS-CoV-2 nucleoprotein binding aptamers were identified and used to develop a Proximity Ligation Assay for sensitive viral protein detection. This assay was further coupled with quantitative PCR to detect both the viral nucleoprotein and viral RNA simultaneously through two fluorescent channels. This dual detection method shows promise for sensitive testing of other viruses. The last chapter focuses on improvements of instrumentation and experimental methodologies. Since CE-MS has its own limitations and drawbacks, improvements had to be made to achieve experimental goals. This chapter describes the development and manufacturing a novel nano sheath liquid interface, allowing for sensitive detection of various compounds. In addition, the chapter describes sample normalization strategies and metabolite identification pipeline. Flaws of CE-MS such as run-to-run variation, as well as susceptibility to matrix effects, needed to be addressed to ensure reproducibility for complex experiments such as metabolomics. Normalization strategy to total UV signal as well as metabolomics pipeline are described in this chapter. In summary, this thesis explores diverse applications of capillary electrophoresis in bioanalysis, from small molecule pharmaceutical analysis to cancer biomarker discovery. It demonstrates the potential of CE in addressing real-world bioanalytical problems and solidifies its versatility.