Mass spectrometry applied to problems in lipid biochemistry: Microchip based approach for lipidomics profiling and analysis of lipid metabolites by LC-MS/MS.

摘要

Lipidomics and metabolomics are powerful tools for the examination of cellular metabolism and physiology. Methods for lipid analysis need to be developed that begin with small samples and do not overly dilute or disperse the sample in the separation process. Microchips provide a platform for interfacing lysis of small cell populations with on-chip solid phase extraction for isolating lipid samples to generate high quality mass spectra from very small samples. Chapter 1 of this dissertation presents a novel method for small scale lipidomics of bacterial cells by microchip based extraction coupled with untargeted profiling of glycerophospholipids using nanoelectrospray ionization mass spectrometry. Chapter 2 and 3 focus on the development of LC-MS/MS methods to study biological pathways. In Chapter 2, I describe a method for analysis of the phospholipids metabolite, GroPIns, in the medium of the pathogenic yeast Candida albicans. This method was applied to aid in the characterization of the GroPIns transport protein, Git1, in C. albicans. Chapter 3 extends the studies of part two and describes an efficient method based on HILIC-MS/MS for the separation and quantification of five lipid-related extracellular metabolites in yeast Saccharomyces cerevisiae. This newly developed methodology was successfully applied to determine the extracellualr levels of glycerophosphoinositol, glycerophosphocholine, glycerol 3-phosphate, inositol and choline in wild type and mutant strains.