Investigation of mass spectrometric techniques for the structural determination and the sequencing of some bacterial capsular polysaccharides from the family Enterobacteriaceae: Klebsiella and Escherichia coli
Creators: name: Lam, Zamas
Publisher: name: University of British Columbia
Year: 2010

description: The structural elucidation of bacterial capsular polysaccharides is traditionally performed by using "wet chemical procedures" but instrumental methods such as nuclear magnetic resonance spectroscopy and novel mass spectrometric techniques are coming into prominence. In this thesis three different mass spectrometric techniques were investigated to establish their applicability for the sequencing of bacterial capsular polysaccharides. These techniques included fast atom bombardment (FAB), desorption chemical ionisation (DCI) and laser desorption ionisation Fourier transform ion cyclotron resonance spectroscopy (LDI-FTICR). The soft ionisation produced by these methods allows sequential loss of individual sugar residues without excess thermal decomposition of the ring. Thus sequencing of oligosaccharides could be achieved. The most common of all these three techniques is FAB which is already considered to be a well established form of soft ionisation, although the exact mechanism of ionisation is unknown. The utilisation of DCI has not been thoroughly exploited in carbohydrate research, due to the non-volatility of oligosaccharides and the possible thermal decomposition of the sample in the source. LDI-FTICR due to the general unavailability of the instrument has only been used for model studies of "shelf carbohydrates". In the course of this work it was found that FAB MS and DCIMS complement each other. The sequence of linear oligosaccharides of up to five sugar units can be deduced from either the native or permethylated sample. If the oligosaccharides investigated were generated by phage-borne enzyme, the total sequence of the native polysaccharide can be established. This was illustrated by the use of FABMS on Klebsiella K44 de-O-acetylated oligosaccharide and the reduced oligosaccharide. The sequence of the polysaccharide was shown to be: [Formula Omitted] The structural elucidation of bacterial capsular polysaccharides is traditionally performed by using "wet chemical procedures" but instrumental methods such as nuclear magnetic resonance spectroscopy and novel mass spectrometric techniques are coming into prominence. In this thesis three different mass spectrometric techniques were investigated to establish their applicability for the sequencing of bacterial capsular polysaccharides. These techniques included fast atom bombardment (FAB), desorption chemical ionisation (DCI) and laser desorption ionisation Fourier transform ion cyclotron resonance spectroscopy (LDI-FTICR). The soft ionisation produced by these methods allows sequential loss of individual sugar residues without excess thermal decomposition of the ring. Thus sequencing of oligosaccharides could be achieved. The most common of all these three techniques is FAB which is already considered to be a well established form of soft ionisation, although the exact mechanism of ionisation is unknown. The utilisation of DCI has not been thoroughly exploited in carbohydrate research, due to the non-volatility of oligosaccharides and the possible thermal decomposition of the sample in the source. LDI-FTICR due to the general unavailability of the instrument has only been used for model studies of "shelf carbohydrates". In the course of this work it was found that FAB MS and DCIMS complement each other. The sequence of linear oligosaccharides of up to five sugar units can be deduced from either the native or permethylated sample. If the oligosaccharides investigated were generated by phage-borne enzyme, the total sequence of the native polysaccharide can be established. This was illustrated by the use of FABMS on Klebsiella K44 de-O-acetylated oligosaccharide and the reduced oligosaccharide. The sequence of the polysaccharide was shown to be: [Formula Omitted] The location of acid-labile pyruvic acid acetal group, like base-labile acetate group, is also difficult to establish chemically. The fragment ions arising from permethylated oligosaccharides were mostly non-reducing end residues. This is due to the stability of oxonium ion formation. However, when an amino sugar was investigated, oxonium ions were not observed. Instead the cleavage took place between the glycosidic oxygen and the reducing end residue. This fragmentation route is in sharp contrast to previously reported spectral data. This may be due to the fact that amino sugars strengthen the glycosidic bond between the oxygen and the carbon-1. LDI-FTICR was investigated for its applicability to a "real" sample. The sequence of the linear Klebsiella K44 de-O-acetylated, phage degraded oligosaccharide was determined from the spectrum. Furthermore, a few positions of linkage were also deduced from ring cleavage fragments. Although linkage positions can be obtained from methylation analysis data, some sugar residues such as deoxyhexoses are more labile than others, thus positions of linkage obtained from LDI-FTICR can be used for confirmation.