Capillary Electrophoresis and Capillary Liquid Chromatography for Analysis of Neurological and Neuroendocrine Signaling

Abstract

Neurological and neuroendocrine disorders result from signaling dysregulation at the molecular, cellular, and multi-cellular levels. This dissertation presents the development of separation methods, using capillary zone electrophoresis (CZE) and capillary liquid chromatography (CLC), for detecting and quantifying small molecules, peptides, and proteins involved in cellular signaling. CZE is a rapid separation technique, making it ideal for monitoring cellular dynamics with high temporal resolution. An ultraviolet - light emitting diode was used for photolytic optical gating of caged fluorophore-labeled biogenic amines, common functional groups in neurotransmitters. Additionally, a novel caged fluorophore with faster reaction kinetics than commercially available dyes was used to label reduced thiols and primary amines in the presence of o-phthalaldehyde. Together this light source and novel caged dye illustrate the utility of these methods for monitoring chemical dynamics during continuous sampling. Many cellular second messengers, including inositol phosphates, are known to exist within the cell, but their dynamics and intermolecular interactions are poorly understood since they lack chromophores or electroactive functional groups making direct detection difficult. Utilizing CZE with capacitive coupled contactless conductivity detection (C4D), biological phosphates were separated and detected based on their high anionic charge, suggesting the utility of C4D in label-free detection of biological molecules. The techniques described above require higher sensitivity to monitor physiologically relevant analyte concentrations; therefore, Hadamard transform capillary electrophoresis (HTCE) was used as a multiplexing method in which multiple separations were performed simultaneously. HTCE resulted in increased sensitivity by decreasing the random background noise. Peptides and proteins propagate signals within or between cells; yet, they are difficult to separate and detect by CZE since their highly charged surfaces result in non-specific adsorption to the capillary wall. To minimize these interactions, stable hybrid phospholipid bilayers were prepared as capillary coatings for CZE separations of cationic proteins. Additionally, stabilized phospholipid bilayer coatings were formed on silica particles through redox polymerization of synthetic, polymerizable lipids. These bilayers were stable after exposure to surfactant, organic solvents, and after storage for one month, suggesting their value as lipid chromatography stationary phases for future incorporation of transmembrane proteins to analyze binding interactions with small molecules.