Year

2013

Degree Name

Doctor of Philosophy

Department

School of Chemistry

Abstract

Lipids serve numerous cellular functions that are essential to the survival of species ranging from the smallest micro-organisms to the largest plants and the most highly evolved animals. In all cases, the ability of an individual lipid (or a class of lipids) to perform its biochemical role is dependent on its molecular structure. As a result, small alterations in the chemical structures of lipids can change their physical and biochemical properties, and thus affect their roles within a living organism. Extensive work on the relationship between lipid bio-activity and molecular structure points to the importance of acyl chain length, relative acyl chain position (i.e., sn-position), chain branching, location and even stereochemistry of carbon-carbon double bonds (i.e., cis versus trans).

It is becoming increasingly apparent therefore, that detailed molecular structure elucidation of lipids is critical to informing our understanding of both their structural and signaling functions. Contemporary lipidomics protocols rely on tandem mass spectrometry and in particular collision-induced dissociation (CID) of even-electron ions. While these approaches are powerful for determining lipid class and identifying the number of carbons and the degree of unsaturation of any acyl chain substituents, they are typically blind to isomeric variants arising from different carbon-carbon bonding motifs within these chains, including double bond position, chain branching and cyclic structures. In this work, we have reviewed the current mass spectrometrybased methods for structural characterisation and investigated some alternative ion activation methods directed toward the complete structure elucidation of lipids.

Ozone-induced dissociation (OzID) was previously developed in our laboratory for localisation of carbon-carbon double bonds by utilising the gas phase ion-molecule reaction between ionised unsaturated lipids and ozone in an ion-trap mass spectrometer. Herein, we evaluated the performance of OzID for both structural elucidation and selective detection of conjugated carbon-carbon double bond motifs within lipids. Strikingly, conjugated lipids were found to react up to one hundred times faster than a comparable non-conjugated isomer for any given metal adduct ion. In addition, ozonolysis of each conjugated isomer was found to yield a unique radical ion that was associated with the position of the conjugated diene motif. This phenomenon has been exploited to undertake neutral-loss (NL) scans on a triple quadrupole mass spectrometer targeting characteristic OzID transitions: giving rise to a new protocol dubbed NLOzID. Herein, we also describe the application of multiple stages of MS utilising different combinations of CID and OzID for structural analysis of glycerolipids, by removing one or two of the esterified fatty acids and leaving the remaining acyl chain(s) for ozonolysis. The method is demonstrated to identify (i) fatty acid position on glycerol backbone and (ii) assignment of the double bond position(s) within a particular acyl chain and assigning the chain to a specific sn-position. It is thus a significant step forward to the complete structural assignment of lipids by mass spectrometry alone.

To identify the structural variations in branched lipids, we have applied radicaldirected dissociation (RDD) to the field of lipidomics for the first time. In this approach, laser irradiation at UV wavelengths (266 nm) is employed to generate lipid radical ions from either non-covalent complexes (i.e., adduct ions) or covalently modified derivatives, both of which contain a photo-caged radical initiator centre. Subsequent activation of the nascent radical ions results in RDD with significant intra-chain fragmentation of acyl moieties. This approach provides diagnostic fragments that are associated with the positions of double bonds and also the positions of chain-branching within individual lipid structures, ranging from simple lipids (e.g., various motifs of fatty acid derivatives) to complex lipids (e.g., glycerophospholipids, sphingomyelins and triacylglycerols). RDD has been used to reveal lipid structural diversity in olive oil and human very-low density lipoprotein. The work described herein demonstrates the utility of several novel ion activation methods in providing structural information on a wide range of lipid species. Taken together, OzID, CID/OzID and RDD represent powerful new tools for contemporary lipidomics.

FoR codes (2008)

0301 ANALYTICAL CHEMISTRY, 0306 PHYSICAL CHEMISTRY (INCL. STRUCTURAL)

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.