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Abstract:
Cells, whether bacterial, fungal or mammalian, are all equipped with metabolic
pathways capable of producing an assortment of structurally and functionally distinct lipid
species. Despite the structural diversity of lipids being recognized and correlated to specific
cellular phenomena and disease states, the molecular mechanisms that underpin this structural
diversity remain poorly understood. In part, this is due to the lack of adequate analytical
techniques capable of measuring the structural details of lipid species in a direct,
comprehensive and quantitative manner. The aim of my thesis study was to establish
methodology for automated and quantitative analysis of molecular lipid species based on
mass spectrometry. From this work a novel high-throughput methodology for lipidome
analysis emerged. The main assets of the methodology were the structure-specific mass
analysis by powerful hybrid mass spectrometers with high mass resolution, automated and
sensitive infusion of total lipid extracts by a nanoelectrospray robot, and automated spectral
deconvolution by dedicated Lipid Profiler software. The comprehensive characterization and
quantification of molecular lipid species was achieved by spiking total lipid extracts with
unique lipid standards, utilizing selective ionization conditions for sample infusion, and
performing structure-specific mass analysis by hybrid quadrupole time-of-flight and ion trap
mass spectrometry. The analytical routine allowed the comprehensive characterization and
quantification of molecular glycerophospholipid species, molecular diacylglycerol species,
molecular sphingolipid species including ceramides, glycosphingolipids and inositolcontaining
sphingolipids, and sterol lipids including cholesterol.
The performance of the methodology was validated by comparing its dynamic
quantification range to that of established methodology based on triple quandrupole mass
spectrometry. Furthermore, its efficacy for lipidomics projects was demonstrated by the
successful quantitative deciphering of the lipid composition of T cell receptor signaling
domains, mammalian tissues including heart, brain and red blood cells, and the yeast
Saccharomyces cerevisiae.
