Doctor of Philosophy
School of Chemistry
Kozlowski, Rachel, Development of mass spectrometry-based methods for the analysis of lipid isomers, Doctor of Philosophy thesis, School of Chemistry, University of Wollongong, 2015. http://ro.uow.edu.au/theses/4454
Glycerophospholipids, or phospholipids, are important biochemical components of many biological organisms. They are involved in many biochemical processes including signal transduction, serving as enzyme substrates for lipoprotein metabolism and serving as ligands for receptors and precursors of essential biomolecules; they are also intracellular traffickers of cholesterol as well as modulators of the immune system. Likely to fulfill these diverse functional roles, not only do phospholipids contain a variety of head groups attached to a glycerol backbone but even more diversity exists within the fatty acyl chain(s) that are attached to the same glycerol backbone. These differences can be very subtle as is the case with phospholipid sn-positional isomers or double bond positional isomers, where the position of the acyl chain(s) attached to the glycerol backbone or the double bond position(s) within these acyl chains can vary only slightly but have vastly different biochemical consequences. Despite this, little is known about the abundances, let alone the biochemical or evolutionary reasons for the existence of these phospholipid isomers in different biological tissues. This paucity of information exists, in part due to the lack of methods that are both specific and sensitive enough to be used to detect and quantify these phospholipid isomers [and others], especially in complex biological extracts. To this end, this thesis describes the development of a number of new analytical workflows that combine ozone-induced dissociation with analytical separations and direct desorption/ionization technologies for the rapid identification of isomeric lipids. The methods developed and described herein have combined liquid chromatography (LC) and desorption electrospray ionization (DESI) with ozonolysis and collision induced dissociation (CID) to develop LC-OzID, LCCID/ OzID and DESI-CID/OzlD. This LC-OzID method has proven successful for the separation, identification and, to an extent, the quantification, of a number of phospholipid double bond positional isomers in complex biological samples. In a similar way, the LC-CID/OzID method described herein has proven successful for the separation and identification and to an extent, quantification, of several phospholipid sn-positional isomers. DESI-CID/OzlD has proven a successful technique for the rapid identification and detection of relative differences in phospholipid sn-positional isomer composition in complex biological extracts spotted on a surface but also sampled directly from tissue. Experimental results from DESI-CID/OzlD experiments also support the likelihood that both complementary phospholipid sn-positional isomers are always present but at varied ratios in different tissues. Preliminary imaging data using DESI-CID/OzlD demonstrate the great potential of this technique for imaging variations in phospholipid sn-positional isomer composition across different regions of a biological tissue.