Mass spectrometric analysis and fingerprint identification of natural lipopolysaccharide vaccine candidates and synthetic liposomal cholesteryl neoglycolipids

El-Aneed, Anas (2006) Mass spectrometric analysis and fingerprint identification of natural lipopolysaccharide vaccine candidates and synthetic liposomal cholesteryl neoglycolipids. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Mass spectrometric fingerprint identification and structural elucidation of natural and synthetic compounds can be accomplished via the use of mass spectrometry techniques namely, electrospray ionization tandem mass spectrometry (ESI-MS/MS) and matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS). In this work, it was opted to use ESI-MS and MALDI-MS, since these are the softest ionization methods which do not require excessive manipulation of the analytes. The identification of MS fingerprints can be effectively used for any quantitative or qualitative studies. -- Lipopolysaccarides (LPS) are poly-sugars enriched on the outer membrane of all gram-negative bacteria, most of which are pathogenic. These sugar complexes are potential candidates with relevance to bacterial vaccine developments. This study established the fragmentation pattern of native LPS extracts isolated from Aeromonas salmonicida, which infects various fish species. The exact molecular structure of lipid A portion and the core region of this bacterium have been precisely established providing evidence for the unreported presence of the reactive phosphate group at the reducing end of the core oligosaccharide. -- LPS moieties (vaccines) have limited antigenic properties when injected alone due to their small size. Therefore a synthetic conjugate of LPS with a protein carrier or the encapsulation of LPS within liposomal carrier will result in the desired antigenic activity. One major limitation for the usage of liposomes is their tendency to aggregate as well as rapid clearance in the circulation system. Synthetic neoglycolipids have been successfully incorporated into liposomal formulations to prolong their half lives as alternative to pegylated liposomes (PEG-liposomes). -- The fragmentation routes of a series of synthetic amphiphilic cholesteryl polyethoxy neoglycolipids were established with the aid of electrospray ionization mass spectrometry with a QqTOF-MS hybrid instrument. The results have shown the unique presence of specific common fingerprints such as [Cholestenet, [(Sugar-spacerOH)+H]⁺, [oxonium]⁺, [oxonium-H₂O]⁺ and, in some cases, [(Cholesterol-spacerOB:)+H]⁺. In addition, the "in situ" formation of an unexpected and unique [Cglycosidet product ion, resulting from an ion-molecule reaction, was observed. This reaction occurs in the collision cell and the ESI interface of the tandem mass spectrometer. Interestingly, this product ion was absent in the case of O-acetylated sugar species.

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