Molecular characterization of the lipidome by mass spectrometry

Abstract

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