Year

2012

Degree Name

Doctor of Philosophy

Department

School of Chemistry

Abstract

Lipids are the main structural component of biological membranes and are important in complex biochemical processes including cellular signalling. Using electrospray ionisation-tandem mass spectrometry (ESI-MS/MS), information regarding fatty acid chain length and degree of unsaturation can be gained for complex lipids. Determining double bond position, however, remains challenging.

To address the limitations of collision-induced dissociation (CID) for structural characterisation, the use of ozonolysis chemistry in conjunction with mass spectrometry was explored. Initial experiments concentrated on performing ozonolysis within the electrospray source of conventional mass spectrometers; referred to here as ozone electrospray ionisation-mass spectrometry (OzESI-MS). In OzESI-MS experiments, two ozonolysis product ions, assigned as an aldehyde and α-methoxyhydroperoxide, are observed from the cleavage of lipid carbon-carbon double bonds. Importantly, the massto- charge ratio of the two ozonolysis product ions allows the unambiguous assignment of double bond position. OzESI-MS was successfully applied to a wide range of lipid standards including glycerophospholipids, sphingomyelins and triacylglycerols, demonstrating the wide utility of OzESI-MS for determining double bond position. While OzESI-MS provides clear information on double bond position for pure, isolated lipids, determining double bond position for lipids present in complex mixtures is difficult and, in many cases, unambiguous assignment of double bond position is not possible. This presents a major limitation to the OzESI-MS technique.

To overcome the problems associated with in-source ozonolysis, instrument modifications were made to a linear ion-trap mass spectrometer in order to allow ozonolysis to be performed on mass-selected lipid ions within the ion trap. Ozonolysis at the site of carbon-carbon double bonds resulted in the formation of two primary ozonolysis product ions, namely an aldehyde and a so-called Criegee ion. This method, referred to as ozone-induced dissociation (OzID), was applied to a wide range of lipid ions including; the deprotonated ions of the acidic phospholipid classes; protonated, lithiated and sodiated ions of phosphatidylcholine (PC) lipids; and sodium adducts of triacylglycerols. As expected, the observed ozonolysis product ions allow the unambiguous assignment of double bond position in all cases. Furthermore, in a significant advancement over OzESI-MS, OzID was successfully applied to lipids from a range of complex biological mixtures.

OzID was also used in conjunction with CID in the MS3 method referred to as CIDOzID. This approach was used to assign double bond position in both the ester and ether-linked chains of an unsaturated ether phosphatidylethanolamine. Moreover, it was found that the [M+Na-183]+ CID product ion from sodiated PC lipids was highly reactive towards ozone. Interestingly, the observed ozonolysis product ions provide further evidence in support of the substitution mechanism proposed by Hsu and Turk for the formation of this ion. In addition, CID-OzID of the sn-positional isomers, PC(16:0/9Z-18:0) and PC(9Z-18:1/16:0), revealed striking differences in product ion distributions that may be used to determine fatty acid sn-position.

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