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. / Science, Faculty of / Chemistry, Department of / Graduate

Call number: LD2668 .T4 1985 M86 / Master of Science

by Lee Man Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 125-137). / Abstract also in Chinese. / THESIS COMMITTEE --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / ABSTRACT (Chinese version) --- p.v / TABLE OF CONTENTS --- p.vi / LIST OF TABLES --- p.xi / LIST OF FIGURES --- p.xv / LIST OF ABBREVIATIONS --- p.xvii / Chapter CHAPTER ONE: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Background of mushrooms --- p.1 / Chapter 1.1.1 --- Cultivated mushrooms --- p.1 / Chapter 1.1.1.1 --- Volvariella volvacea --- p.3 / Chapter 1.1.1.2 --- Pleurotus sajor-caju --- p.4 / Chapter 1.1.1.3 --- Pleurotus tuber-regium --- p.5 / Chapter 1.1.2 --- Chemical composition and nutritional value --- p.6 / Chapter 1.1.3 --- Composition and structure of fungal cell wall --- p.9 / Chapter 1.1.4 --- Medicinal attributes of β-glucan in mushrooms --- p.10 / Chapter 1.1.5 --- Structure and antitumor activity β-glucan --- p.11 / Chapter 1.2 --- Dietary fiber --- p.14 / Chapter 1.2.1 --- Composition of dietary fiber --- p.14 / Chapter 1.2.2 --- Preparation of dietary fiber --- p.18 / Chapter 1.3 --- Structural analysis of polysaccharides --- p.19 / Chapter 1.3.1 --- Isolation of polysaccharides --- p.19 / Chapter 1.3.2 --- Methylation analysis --- p.20 / Chapter CHAPTER TWO : --- MATERIALS AND METHODS --- p.22 / Chapter 2.1 --- Sources and preparation of mushroom samples --- p.22 / Chapter 2.1.1 --- V. volvacea --- p.22 / Chapter 2.1.2 --- P. sajor-caju --- p.22 / Chapter 2.1.2.1 --- Fungal strain --- p.22 / Chapter 2.1.2.2 --- Production of spawn --- p.22 / Chapter 2.1.2.3 --- Production of fruiting bodies --- p.23 / Chapter 2.1.3 --- p. tuber-regium --- p.23 / Chapter 2.2 --- Analysis of mushroom composition --- p.24 / Chapter 2.2.1 --- Moisture content --- p.24 / Chapter 2.2.2 --- Starch content --- p.24 / Chapter 2.2.2.1 --- Total glucose --- p.24 / Chapter 2.2.2.2 --- Free glucose --- p.25 / Chapter 2.2.2.3 --- Measurement of glucose content --- p.25 / Chapter 2.2.2.4 --- Total starch content --- p.25 / Chapter 2.2.3 --- Crude protein content --- p.26 / Chapter 2.2.4 --- Amino acid analysis --- p.27 / Chapter 2.3 --- Preparation of mushroom fiber material --- p.28 / Chapter 2.3.1 --- Enzymatic method --- p.28 / Chapter 2.3.1.1 --- Total dietary fiber (TDF) --- p.28 / Chapter 2.3.1.2 --- Insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) --- p.29 / Chapter 2.3.2 --- Chemical method --- p.29 / Chapter 2.3.2.1 --- Cell wall material --- p.29 / Chapter 2.4 --- Chemical composition of mushroom fiber material --- p.31 / Chapter 2.4.1 --- Monosaccharide composition of non-starch polysaccharides (NSP) --- p.31 / Chapter 2.4.1.1 --- Acid depolymerisation --- p.31 / Chapter 2.4.1.2 --- Neutral sugar derivatization --- p.31 / Chapter 2.4.1.3 --- Determination of neutral sugars by gas chromatography (GC) --- p.32 / Chapter 2.4.1.4 --- Uronic acid content --- p.32 / Chapter 2.4.2 --- Resistant starch content --- p.33 / Chapter 2.4.3 --- Residual protein content --- p.34 / Chapter 2.5 --- Fractionation of mushroom fiber material --- p.34 / Chapter 2.5.1 --- Solvent extraction --- p.34 / Chapter 2.5.2 --- Anion-exchange chromatography --- p.35 / Chapter 2.5.3 --- Gel permeation chromatography --- p.36 / Chapter 2.6 --- Structural analysis of mushroom fiber material --- p.37 / Chapter 2.6.1 --- Linkage analysis by methylation --- p.37 / Chapter 2.6.1.1 --- Preparation of methylsufinyl carbanion (Dimsyl) --- p.37 / Chapter 2.6.1.2 --- Preparation and dissolution of sample --- p.37 / Chapter 2.6.1.3 --- Methylation --- p.38 / Chapter 2.6.1.4 --- Hydrolysis --- p.38 / Chapter 2.6.1.5 --- Reduction and acetylation --- p.39 / Chapter 2.6.1.6 --- Determination of partially methylated alditol acetate (PMAA) by gas chromatograph-mass spectrometry (GC-MS) --- p.39 / Chapter 2.6.2 --- Fourier-transform infrared (FTIR) spectroscopy --- p.40 / Chapter CHAPTER THREE : --- RESULTS AND DISCUSSION --- p.41 / Chapter 3.1 --- Chemical composition of mushrooms --- p.41 / Chapter 3.1.1 --- Moisture content --- p.41 / Chapter 3.1.2 --- Carbohydrate content --- p.41 / Chapter 3.1.3 --- Protein content --- p.44 / Chapter 3.1.4 --- Amino acid profile --- p.44 / Chapter 3.1.5 --- Dietary fiber content --- p.48 / Chapter 3.1.6 --- Cell wall material --- p.53 / Chapter 3.1.7 --- Comparison of the yield and composition of TDF and CWM --- p.55 / Chapter 3.1.8 --- "Monosaccharide composition of the dietary fiber (TDF, IDF and SDF) and cell wall material (CWM)" --- p.57 / Chapter 3.2 --- Fractionation of TDF and CWM --- p.69 / Chapter 3.2.1 --- Solvent extraction --- p.69 / Chapter 3.2.2 --- Monosaccharide composition of solvent fractionated TDF and CWM --- p.71 / Chapter 3.2.3 --- Anion-exchange chromatography --- p.78 / Chapter 3.2.4 --- Gel permeation chromatography --- p.82 / Chapter 3.2.5 --- Monosaccharide composition of fractionated fiber material by anion-exchange chromatography --- p.84 / Chapter 3.3 --- Structural analysis --- p.86 / Chapter 3.3.1 --- Partially methylated alditol acetate (PMAA) --- p.86 / Chapter 3.3.1.1 --- Alkali-extracted water-soluble fractions of V. volvacea fiber material --- p.95 / Chapter 3.3.1.2 --- Alkali-extracted water-soluble fractions of P. sajor-caju fiber material --- p.99 / Chapter 3.3.1.3 --- Alkali-extracted water-soluble fractions of P. tuber-regium fiber material --- p.102 / Chapter 3.3.1.4 --- Alkali-extracted water-insoluble fractions of the mushroom fiber material --- p.106 / Chapter 3.3.1.5 --- Alkali- and acid- resistant fractions of the mushroom fiber material --- p.109 / Chapter 3.3.2 --- Infrared spectroscopy --- p.112 / Chapter 3.4 --- "β (l→3), (→4) glucan" --- p.119 / Chapter CHAPTER FOUR : --- CONCLUSION --- p.121 / REFERENCES --- p.125 / RELATED PUBLICATIONS --- p.137

Chan Pui Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 98-104). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.i / LIST OF FIGURES --- p.iv / LIST OF TABLES --- p.vii / ABBREVIATIONS --- p.viii / Chapter Chapter one --- Research Background / Chapter 1.1 --- Carbohydrates --- p.2 / Chapter 1.2 --- Impact of molecular weight of polysaccharides --- p.5 / Chapter 1.3 --- Molecular Weight Determination of polysaccharides --- p.6 / Chapter 1.3.1 --- Laser Scattering --- p.6 / Chapter 1.3.2 --- Gel Permeation Chromatography --- p.7 / Chapter 1.3.3 --- Mass spectrometry --- p.9 / Chapter 1.4 --- Matrix assisted laser desorption/ ionization (MALDI) --- p.10 / Chapter 1.4.1 --- Laser desorption --- p.10 / Chapter 1.4.2 --- Matrix-assisted laser desorption / ionization (MALDI) --- p.11 / Chapter 1.5 --- MALDI-TOFMS analysis of polymers --- p.14 / Chapter 1.6 --- Outline of the present work --- p.16 / Chapter Chapter two --- Experimental and Instrumentation / Chapter 2.1 --- Matrix-assisted laser desorption/ ionization Time of flight Mass Spectrometry (MALDI-TOFMS) --- p.18 / Chapter 2.2 --- Delayed extraction --- p.20 / Chapter 2.3 --- Time of flight mass spectrometry (TOFMS) --- p.20 / Chapter 2.3.1 --- Linear time-of-flight mass spectrometry --- p.20 / Chapter 2.3.2 --- Reflectron --- p.21 / Chapter 2.4 --- Instrumentation --- p.23 / Chapter 2.4.1 --- Laser system --- p.24 / Chapter 2.4.2 --- Ion source --- p.26 / Chapter 2.4.3 --- Ion deflection --- p.26 / Chapter 2.4.4 --- Detection --- p.27 / Chapter 2.4.5 --- Reflector --- p.27 / Chapter 2.4.6 --- Data acquisition --- p.29 / Chapter 2.5 --- Experimental --- p.29 / Chapter 2.5.1 --- Sample preparation --- p.29 / Chapter 2.5.2 --- Calibration --- p.33 / Chapter 2.6 --- Data analysis --- p.33 / Chapter Chapter three --- Use of ammonium fluoride as co-matrix / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Results and discussion --- p.37 / Chapter 3.2.1 --- Effect of co-matrix --- p.45 / Chapter 3.2.2 --- Effect of sample preparation --- p.49 / Chapter 3.2.3 --- Analysis of dispersed dextran --- p.52 / Chapter 3.3 --- Conclusion --- p.55 / Chapter Chapter four --- Effect of sample preparation / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Experimental --- p.57 / Chapter 4.2.1 --- Sample preparation --- p.57 / Chapter 4.3 --- Results and discussion --- p.59 / Chapter 4.4 --- Conclusion --- p.71 / Chapter Chapter five --- Development of liquid matrix systems / Chapter 5.1 --- Introduction --- p.73 / Chapter 5.2 --- Experimental --- p.75 / Chapter 5.2.1 --- Sample preparation --- p.75 / Chapter 5.3 --- Results and discussion --- p.76 / Chapter 5.3.1 --- Formulation of matrix solutions --- p.76 / Chapter 5.3.2 --- Use of liquid matrix system --- p.87 / Chapter 5.3.3 --- Analysis of dispersed dextran --- p.90 / Chapter 5.4 --- Conclusion --- p.93 / Chapter Chapter six --- Conclusion / Chapter 6.1 --- Conclusion --- p.95 / References --- p.98 / Appendix / Appendix 1 Chemical structure of matrices / Appendix 2 Chemical structure of solubilizing agents / Appendix 3 Chemical structure of liquid supports / Appendix 4 Chemical structure of additives