Tandem mass spectrometric analysis of a series of bacterial lipid as belonging to the Vibrionaceae family

Almostafa, Mervt (2017) Tandem mass spectrometric analysis of a series of bacterial lipid as belonging to the Vibrionaceae family. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

This study presents the mass spectrometry structural elucidations of the extracted heterogeneous lipid As mixtures, obtained from native lipopolysaccharides (LPSs) isolated from different types of the Vibrionaceae family. As this Gram-negative bacteria family is well known to cause many serious animal diseases and human diseases. This investigation was originally aimed to investigate the treatment of bacterial infected fish with phages as a potential treatment for pathogenic bacterial infections. In this thesis, I shall discuss the electrospray ionization mass spectrometry (ESI-MS) and low-energy collision induced dissociation tandem mass spectrometry analysis (CID-MS/MS); and MALDI-TOF-MS/MS (high-energy CID-MS/MS) of a heterogeneous mixture of lipid As. These lipids As were isolated from the rough lipopolysaccharide (LPS) of the mutant wild strain of the Gram-negative bacteria Aeromonas. liquefaciens SJ-19, Aeromonas. hydrophilla SJ-55, and Aeromonas. salmonicidia SJ-113. These strains of bacteria were grown in the presence of phages and were isolated by the aqueous phenol method from this series of LPS of the rough wild strain of Gram-negative bacteria. Hydrolysis of the different LPS was accomplished with 1% acetic acid, and purification was by chromatography (Gel filteration affinity) using Sephadex G-50 and SephadexG-15. These MS and MS/MS complex structural investigation were accomplished via the use of different hybrid MS instruments such as: ESI-MS with a fourier transform ion cyclotron (FTICR), a triple quadrupole (QqQ), and MALDI-TOF/TOF-MS instruments. In that respect, low- and high-energy CID-MS/MS (known as tandem-in-space) and FTICR-MS/MS (known as tandem-in-time) mass spectrometry analyses were chosen for iii the structural elucidation of this different series of lipid As isolated from A. liquefaciens SJ-19, hydrophilla SJ-55, and salmonicidia SJ-113a. Excellent elucidation structural results were obtained by using low collision induced (CID-MS/MS) analyses of the various heterogeneous mixtures of lipid As precursor deprotonated molecules with the two different triple quadrupole (QqQ) and FTICR-MS/MS instruments. The concomitant uses of high-energy CID-MS/MS obtained with the MALDI-TOF/TOF-MS instrument allowed the further elucidation of these complex biomolecules and can be effectively used for any structural identifaction studies. Identifying the exact structures of these microheterogenious lipid As, obtained from Gram-negative bacteria grown in presence of phages, were by using mass spectrometry and tandem mass spectrometry techniques using different MSMS instruments, allowed to conclude that the biosynthesis of these series of mutant rough LPSs were indeed affected and produced a heterogeneous mixtures of different lipid As. Finally, this structural elucidation study provided us with a better understanding of the stereospecific gas-phase mass spectral fragmentation pathways; also, the exact sequence of these series of lipid A obtained from the rough bacteria LPSs, which have not yet been precisely established.

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