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

A completely automated mass spectrometer system has been developed to measure the oxygen isotope ratio of carbon dioxide samples. The system has been shown to have a precision of 0.03°/oo, which is comparable to that quoted for any other system in the world. In addition, the facility is capable of analyzing over one hundred samples per day. The system uses an Interdata minicomputer as the primary controller. The minicomputer monitors the quality of analyses, on-line, and thereby insures that all DEL values are measured to at least 0.04°/oo. Host of the sophistication resides in intelligent controllers within the mass spectrometer console. This design gives a technician considerable power when operating the system in a manual mode. The intelligence of the system is contained within hardware circuits, software within the minicomputer and firmware written for a Motorola 6802 microprocessor. A major contribution of this thesis has been the design and installation of an automated mass spectrometer inlet system. A microprocessor based inlet system controller maximizes the throughput of carbon dioxide samples within the inlet system. The inlet system normally contains four different aliquots of carbon dioxide and introduces these samples to the mass spectrometer, in proper sequence, through a single mass spectrometer admittance leak. The system has been used in the analysis of 111 samples of ice taken from the Steele Glacier, Yukon Territory. The samples taken from a vertical borehole, displayed a sawtooth variation of the oxygen isotope ratio with depth. The data have been explained by a physical model described in an appendix to this thesis. If our interpretation is correct, the isotopic variations have recorded at least four surges of the Steele Glacier. / Science, Faculty of / Earth, Ocean and Atmospheric Sciences, Department of / Graduate

Operational characteristics and performance of two mass spectrometers incorporating improved versions of the Very High Yield Electron Impact (VHY-EI) ion source are described. The ionizers are designed around a large volume, low pressure, hot cathode Penning geometry and operate at source pressures of 10⁻⁵ torr and below. In combination with low spherical aberration focusing optics, mass analyzed ion currents 100 to 1000 times more intense than those produced by conventional ionizers operated under identical or comparable conditions of resolution, analyzer type, sample pressure, and electron impact energy are obtained. In addition, high ionization-mass analysis efficiencies have been demonstrated for a variety of organic compounds with sample mass flow rates approaching 1 mg/min. Typical electron impact spectra are obtained with no evidence of pyrolysis or ion molecule reactions. Unusually intense spectra can be produced with low energy electrons.

This thesis focuses on analysis of reaction products of phthalaldehyde with selected amino acids using the combination of high-performance liquid chromatography with mass spectrometry. Only one product with dihydroisoindole structure is formed in the case of simple amino acids (glycine, glycine ethyl ester, alanine, α-aminobutyric acid, valine, leucine and isoleucine). Reactions of phthalaldehyde with amino acids with two amino groups (lysine, asparagine, glutamine and arginine) yield different types of compounds. Main products are formed by the interaction of both of the amino groups with one molecule of phthalaldehyde. Apart from this type of product, these reactions also result in formation of the expected analogous structures of dihydroisoindoles. Moreover, the formation of products containing one molecule of amino acid and two molecules of phthalaldehyde is not excluded. The products were structurally analysed by the use of high-performance liquid chromatography and tandem mass spectrometry. Reaction products of phthalaldehyde with α-aminobutyric acid, valine, leucine, isoleucine and lysine were isolated and further analyses such as electron ionisation with double focusing sector analyser and nuclear magnetic resonance were performed. Pieces of information resulting from the analysis of...

Understanding of molecular events involved in food safety and human health has become a major concern of contemporary life. Mass spectrometry (MS) is a powerful tool for characterization of complex food ingredients and biological molecules. Advances in MS-based techniques have offered new opportunities to understand the chemical changes occurring during food storage and processing as well as the molecular events perturbed by either endogenous or exogenous stimulus. In this thesis, we developed novel MS-based approaches for authentication of edible oil (i.e., edible vegetable oil, deep frying oil and gutter oil), assessment of genotoxicity of fatty acid hydroperoxides, and investigation of metabolic deregulation in pleural effusion and tissue of human lung cancer, aiming to gain a better understanding of food safety and human health. MS-based methods were developed for authentication of edible vegetable oil adulterated with used cooking oil. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that six monoglycerides could be used as the markers to discriminate the used cooking oil (e.g., deep frying oil and gutter oil) and qualified edible oil. Accumulation behavior of these six monoglycerides was detected in the repeatedly heated edible oils. Quantitation of the monoglycerides enabled authentication of commercial olive oil adulterated with a small amount of used cooking oil (approximately 1%). In addition, a matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method was developed for visual authentication of edible oil samples, including commercial vegetable oil, used cooking oil and adulterated edible oil. The method provided the capability for quantifying major chemical composition of edible oil, such as triglycerides, diglycerides and monoglycerides. The present method required minimal sample preparation and allowed screening of oil samples with high throughput (approximately 360 samples per day), providing a simple way to authenticate different types of oil samples. Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was applied to investigate the genotoxicity of two fatty acid hydroperoxides, i.e., 13-hydroperoxyoctadeca-cis-9,trans-11-dienoic acid (13-HPODE) and 13-hydroperoxy-9,11E,15Z-octadecatrienoic acid (13-HPOTE), which are important inducers of oxidative deterioration in oils and fats generated from the oxidation of linolenic acid and linoleic acid. The results demonstrated that the α,β-unsaturated aldehydes, such as 4-oxo-2-nonenal, dioxo-10-dodecenoic acid, 4-hydroperoxy-2-nonenal and 4-hydroxy-2-nonenal are the main degradation compounds of 13-HPODE and 13-HPOTE, which can covalently bound to deoxyribose-nucleosides and ribose-nucleosides to form adducts. This study provided evidences regarding the genotoxicity of fatty acid hydroperoxides at the molecular level. MS-based metabolomics methods were developed and applied for the investigation of metabolic signatures of pleural effusion and tissue of human lung cancer. A database-assisted global metabolomics method was established with utility of LC-Orbitrap MS, followed by automated mass spectral searching. The method enabled unbiased identification of 194 endogenous metabolites in pleural effusions caused by tuberculosis and malignancy. Among which, 33 differential metabolites involved in tryptophan catabolism, bile acid biosynthesis, and β-oxidation of fatty acids were found between tuberculous and malignant pleural effusions, which provided non-invasive biomarkers for diagnosis of pleural effusion samples with high sensitivity and specificity. In addition, a large-scale targeted metabolomics method was developed, which enabled reliable detection of over 400 biological metabolites, covering 92 metabolic pathways in human samples. The method was applied to characterize the metabolic profiles of non-small cell lung cancer (NSCLC) tissues. A number of distant metabolic pathways were found to be differentiated between tumor and normal tissues of lung squamous cell carcinoma and adenocarcinoma, including purine metabolism, citric acid cycle, amino acid metabolism, urea cycle, and ammonia recycling. In addition, several metabolites, such as adenosine, glutamate, glucose 1,6-bisphosphate, betaine, creatine and methionine sulfoxide were found to be associated with prognosis of NSCLC patients, which might provide potential biomarkers to monitor metabolic characteristics of NSCLC patients and treatment outcomes of the cancer.

Understanding of molecular events involved in food safety and human health has become a major concern of contemporary life. Mass spectrometry (MS) is a powerful tool for characterization of complex food ingredients and biological molecules. Advances in MS-based techniques have offered new opportunities to understand the chemical changes occurring during food storage and processing as well as the molecular events perturbed by either endogenous or exogenous stimulus. In this thesis, we developed novel MS-based approaches for authentication of edible oil (i.e., edible vegetable oil, deep frying oil and gutter oil), assessment of genotoxicity of fatty acid hydroperoxides, and investigation of metabolic deregulation in pleural effusion and tissue of human lung cancer, aiming to gain a better understanding of food safety and human health. MS-based methods were developed for authentication of edible vegetable oil adulterated with used cooking oil. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that six monoglycerides could be used as the markers to discriminate the used cooking oil (e.g., deep frying oil and gutter oil) and qualified edible oil. Accumulation behavior of these six monoglycerides was detected in the repeatedly heated edible oils. Quantitation of the monoglycerides enabled authentication of commercial olive oil adulterated with a small amount of used cooking oil (approximately 1%). In addition, a matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method was developed for visual authentication of edible oil samples, including commercial vegetable oil, used cooking oil and adulterated edible oil. The method provided the capability for quantifying major chemical composition of edible oil, such as triglycerides, diglycerides and monoglycerides. The present method required minimal sample preparation and allowed screening of oil samples with high throughput (approximately 360 samples per day), providing a simple way to authenticate different types of oil samples. Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was applied to investigate the genotoxicity of two fatty acid hydroperoxides, i.e., 13-hydroperoxyoctadeca-cis-9,trans-11-dienoic acid (13-HPODE) and 13-hydroperoxy-9,11E,15Z-octadecatrienoic acid (13-HPOTE), which are important inducers of oxidative deterioration in oils and fats generated from the oxidation of linolenic acid and linoleic acid. The results demonstrated that the α,β-unsaturated aldehydes, such as 4-oxo-2-nonenal, dioxo-10-dodecenoic acid, 4-hydroperoxy-2-nonenal and 4-hydroxy-2-nonenal are the main degradation compounds of 13-HPODE and 13-HPOTE, which can covalently bound to deoxyribose-nucleosides and ribose-nucleosides to form adducts. This study provided evidences regarding the genotoxicity of fatty acid hydroperoxides at the molecular level. MS-based metabolomics methods were developed and applied for the investigation of metabolic signatures of pleural effusion and tissue of human lung cancer. A database-assisted global metabolomics method was established with utility of LC-Orbitrap MS, followed by automated mass spectral searching. The method enabled unbiased identification of 194 endogenous metabolites in pleural effusions caused by tuberculosis and malignancy. Among which, 33 differential metabolites involved in tryptophan catabolism, bile acid biosynthesis, and β-oxidation of fatty acids were found between tuberculous and malignant pleural effusions, which provided non-invasive biomarkers for diagnosis of pleural effusion samples with high sensitivity and specificity. In addition, a large-scale targeted metabolomics method was developed, which enabled reliable detection of over 400 biological metabolites, covering 92 metabolic pathways in human samples. The method was applied to characterize the metabolic profiles of non-small cell lung cancer (NSCLC) tissues. A number of distant metabolic pathways were found to be differentiated between tumor and normal tissues of lung squamous cell carcinoma and adenocarcinoma, including purine metabolism, citric acid cycle, amino acid metabolism, urea cycle, and ammonia recycling. In addition, several metabolites, such as adenosine, glutamate, glucose 1,6-bisphosphate, betaine, creatine and methionine sulfoxide were found to be associated with prognosis of NSCLC patients, which might provide potential biomarkers to monitor metabolic characteristics of NSCLC patients and treatment outcomes of the cancer.

The aryl hydrocarbon nuclear translocator (ARNT) belongs to the family of basic helix loop helix proteins. ARNT forms a heterodimer with aryl hydrocarbon receptor (AhR). This heterodimer binds to the dioxin responsive element (DRE) causing the regulation of the gene expression of some enzymes such as CYP1Al. Studies show that the ARNT-AhR heterodimer needs protein factors to bind to DRE, and most of these protein factors are still unknown. ARNT also heterodimerizes with hypoxia inducible factor 1 a (HIF-1 a) which mediates the cellular responses to hypoxia. The purpose of this research is to use a combination of affinity chromatography and mass spectrometry techniques to identify the proteins that interact with ARNT. We chose mouse liver as the protein source. We planned to isolate the ARNT interacting proteins from the mouse liver protein extract by using the TALON® resin column bounded with 6x HIS-ARNT. Two negative control experiments were done. The first one by the application of the liver extract only on non-ARNT bound column. The second one by the application of 100 mg of bovine serum albumin on 6x HIS-ARNT bound column. The mouse liver extract was applied on 6x HIS-ARNT bound column. The column was washed with an increasing concentration of potassium chloride (0.05 M- 1 M). ARNT was eluted with a buffer containing 250 and 500 mM imidazole. The different washing fractions were compared with the negative control experiments. There was no difference between this and experiment and negative controls. We also tested using in-vitro chemical cross-linking with formaldehyde. Some distorted bands that may '· represent crosslinked proteins appeared above ARNT molecular weight by the addition of 1% paraformaldehyde for 20 minutes at 37°C, and for 2 hours at 30° and room temperatures. These bands were absent in the negative control experiments. The mass spectrometric protocols for identification of trace amount of proteins using peptide mass fingerprinting were tested using a standard protein (Bovine Serum Albumin).

<p dir="ltr">In mass spectrometry, analyte molecules are ionized by various mechanisms, such that they can be manipulated by electric and magnetic fields. By performing such manipulations, the mass to charge (<i>m/z</i>) ratio of the analyte molecules can be measured. Many methods for ion manipulation that allow for such <i>m/z </i>determination have been developed in the form of different mass analyzers. These include but are not limited to magnetic/electric sector instruments, time-of-flight (TOF) mass analyzers, 3D quadrupole ion traps, quadrupole mass filters, linear quadrupole ion traps (LITs), Fourier-transform ion cyclotron resonance (FT-ICR) instruments, Orbitrap mass analyzers, and electrostatic linear ion traps (ELITs). Each of these mass analyzers has unique advantages and disadvantages resulting from its specific mechanism of operation, allowing each of these to find a niche in mass spectrometry applications. The ELIT is a mass analyzer composed of two opposing ion mirrors, which cause trapped high energy ions to oscillate along a linear axial trajectory. The oscillation period of ions in the ELIT is dependent on injection energy, the potential energy and electrode geometry of the ELIT, and the ion <i>m/z</i> ratio. As such, mass spectra can be measured by measuring the frequency of ion packets in the ELIT in Fourier-transform (FT) operation mode, or by or by allowing ions to separate spatially in an <i>m/z </i>dependent manner before detection via a microchannel plate (MCP) in multiple-reflection time-of-flight (MR-TOF) mode of operation. The ability to perform two orthogonal mass analysis techniques in a single mass analyzer is one key advantage of the ELIT. Both FT-MS, and MR-TOF mode of operation are high-resolution techniques, making the ELIT unique in its excellent performance characteristics despite low complexity and manufacturing cost. Additionally, the ELIT can be used to perform high-resolution ion isolations, which makes it especially attractive for tandem-MS.</p><p dir="ltr">In chapter 1, the operating principles for MR-TOF and FT-MS modes of operation in an ELIT are discussed. In chapter 2 and 3, the performance, limitations, and applications of the mirror-switching isolation technique in the ELIT are discussed. Given the high-resolution performance of the ELIT for both mass analysis and ion isolation, it is clear that the ELIT has great potential for tandem-MS applications which require high-resolution in either the precursor selection, mass analysis step, or both. In chapters 4, 5, and 6, the implementation and development of infrared multi-photon dissociation, and surface-induced dissociation techniques in the ELIT are discussed, and it is shown that the ELIT can be used to as a standalone tandem mass spectrometer. While not performed on the ELIT instrument, the charge-based valet parking technique discussed in chapter 7 applies to tandem-MS as a whole, as it is shown to improve fragment yield in ETD. Finally, in chapter 8, the future directions of development for the ELIT mass analyzer are discussed.</p>

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