Wheat is the most agronomically important crop with an annual production of approximately 680 million tonnes per year over the five year period of 2008-2012 (Shewry and Tatham 2016). Wheat typically contributes about 20% of the total calorie intake in Western Europe and between 50-70% in some countries in North Africa and in West and Central Asia. It is estimated that in order to meet the continuous growing global demand wheat production needs to increase by 50% by 2050. Wheat is most commonly consumed as bread, pasta and noodles however it is also used as a food ingredient in other types of foods such as sauces and condiments. The versatility of wheat is largely determined by the unique physiochemical properties of gluten (Bailey 1941). Gluten is one of the earliest proteins to be studied, and was first described by Beccari in 1728 (Bailey 1941) and is readily isolated from wheat flour as a viscoelastic mass. Gluten is a complex mixture of proteins which are the major seed storage proteins found in the cereal grains wheat, barley, rye and oats. Gluten accounts for 70-80% of the total protein content in wheat grains and is traditionally divided into two groups based on their solubility called gliadins and glutenins (Osborne 1907). In genetically pre-disposed patients gluten is able to elicit a non-IgE mediated T-cell response known as coeliac disease (CD). CD affects approximately 1% of the global population for which there is no cure. As no cure is available patients must adhere to a strict gluten-free diet which is often costly and socially excluding. The Codex Standard states that gluten-free foods must contain less than 20 ppm of gluten from wheat, barley, rye and oats and their crossbreeds (FAO/WHO 1983). The Codex Standard also recommends using immunobased methods (or alternative methods) that are able to achieve appropriate sensitivity and specificity for the detection and quantification of gluten with a 10 ppm limit of detection (FAO/WHO 1983). Consequently the current gold standard method for detection of gluten is enzyme linked immunosorbent assay (ELISA) utilising the R5 antibody, however this method is not without shortcomings. Proteomics by mass spectrometry has the potential to offer an alternative, complementary method to determine gluten proteins in foods but for the methodology to become fully validated and accepted it must also overcome similar challenges to immunoassay methods, such as effective extraction of samples and the identification of peptide targets with the requisite specificity. In this research a global approach is taken to aid the development of gluten detection methods using mass spectrometry. One of the major hurdles that has stunted the development of mass spectrometry methods for the detection and quantification for gluten is the lack of protein sequence databases which are required to undertake the MS data searching. In the first results chapter of the thesis a curated gluten protein sequence database was developed (GluPro), and investigated for its utility as a MS data searching tool. It was observed that utilising the GluPro database resulted in improved protein identifications. Following the development of the curated database, extensive method development was carried out to undertake the most extensive background characterisation of the gluten proteome to date using discovery proteomics. To ensure the most comprehensive profile was obtained a number of extraction protocols were investigated and two mass spectrometry platforms with intrinsic differences utilising different modes of acquisition were used. This resulted in the most comprehensive profile of the gluten proteome to date being obtained. In order to meet the continually growing global demand for wheat previous mentioned it is considered that this may be done through the use of genetically modified crops with improved traits such as pesticide resistance. Resulting in the very real possibility of GM crops being introduced into the food supply chain, however there is much widespread public concern regarding the toxicity and allergenicity of genetically modified crops. As wheat is already listed as one of the major eight allergens, it is crucial to be able to undertake safety assessments which are able to assess the toxicity and allergenicity and determine if the GM crop is substantially equivalent to the non-GM counterpart. In the third part of this thesis it is shown how the MS method developed in the previous chapters could be applied and has great potential to be used for safety assessment. Further to this, it is demonstrated how utilising the additional information gathered during the curation of the GluPro database was able to ground the results into in silico measure of toxicity. In the final part of this research all information gathered was interrogated to pick appropriate MRM target peptides, which were unique to a single gluten protein and reproducibly observed to be free from modification. The peptides were synthesised with heavy labels to develop a targeted method to replace current ELISA methods for the detection and quantification. Unexpectedly mass shifts were observed for the precursor ion corresponding to deamidation of the synthetic peptides. Further investigation was undertaken to understand the location and cause of the deamidation sites. This development leads into further recommendation for future development of MRM methods for the detection and quantification of gluten.

Elemental detection is an emerging area in bioanalysis. Thanks to the rapid advancement in instrumentation such as inductively coupled plasma-mass spectrometry (ICP-MS), low detection limit and quick analysis can be achieved. Besides, ICP-MS also suffers less matrix effect as compared to molecular mass spectrometry, so a precise and accurate detection of toxic or essential elements can be provided. Different types of sample introduction or separation systems such as laser ablation (LA) and liquid chromatograph (LC) are excellent hyphenation options for the elemental detection apart from the total analysis of standalone ICP-MS analysis. Spatial analysis and speciation of the two mentioned techniques provide additional merits to the elemental detection in bioanalysis.;LA-ICP-MS makes use of a laser to ablate the solid sample, and the generated sample aerosol is then transferred to ICP-MS for detection. It can be used for bioimaging. There are examples of LA mapping of biological tissues to reveal the spatial distribution of metal, to study the neurodegenerated disease in brain or the accumulation in metallodrug in tumor mass. In order to incorporate the imaging tool in drug development, in the first part of this thesis, LA-ICP-MS bioimaging of liver and kidney was performed to compare the differential spatial distribution of two structurally different platinum-based anti-cancer drug candidates. It was expected that this approach can assist the chemical modification in drug development.;To put this idea a step further, the spatial analysis tool was tested for its potential in therapeutic drug monitoring. Hair profiling in whiskers of mice treated with vanadium anti-diabetic complex or gadolinium-based contrasting agents at different dosage levels were conducted. Results shown that different deposition behaviors and accumulation/elimination profile can be observed, demonstrating a great potential in routine clinical application.;On the other hand, LC-ICP-MS offers the possibility for speciation study. Several accessories for organic solvent introduction in ICP-MS make the coupling of reverse phase chromatography using high percentage of organic solvent in the mobile phase more convenient. To demonstrate the advantage of this configuration, a speciation of bromine-containing drug in mice urine and plasma was included in the last part of this project for metabolite profiling study.;In Short, this work presents several useful hyphenated techniques of ICP-MS in bioanalysis, proving the tremendous potential of elemental detection in drug development (assisting molecular modification in drug design and metabolite profiling) and therapeutic drug monitoring (hair profiling)

The analysis and quantitation of neurologically-relevant molecules requires detection methods that are sensitive, selective, and applicable to a wide range of molecules. Targeted analysis using tandem mass spectrometry allows for the detection of molecules from complex matrices with an added level of selectivity. Mass spectrometry is on the leading edge of technological advances and improvements in our understanding of the intricate workings of the brain, allowing us to develop better models and better therapeutic approaches. In this thesis, I use tandem mass spectrometry to investigate two classes of neurochemicals: classical neurotransmitters, and potential therapeutic drugs based on endogenous neuropeptides. Chapter 1 will introduce existing sampling techniques and detection schemes for small molecule neurotransmitters and small peptides. We will also introduce two key concepts: insect models for understanding human neurotransmission, and the role of the blood-brain barrier in developing CNS-active pharmaceuticals. In Chapter 2 we develop a method to quantify small molecule neurotransmitters in tissue homogenate for the purpose of understanding how the bulk content of an insect brain can change under differing circumstances. Our approach allows for the analysis of a wider range of compounds with improved throughput compared to existing methods. Chapter 3 expands this method for the quantitation of five biogenic amines in Apis mellifera, to investigate the effect of infection by the microsporidian Nosema ceranae. Chapter 4 explores the role of glycosylation on the stability and blood-brain barrier permeability of peptide-based drugs. Chapter 5 expands this work to a series of Angiotensin 1-7 derivatives, for a study of the effect of different structural modifications to peptide-based drugs, with the goal of driving drug development toward more effective pharmaceuticals. Chapter 6 concludes this work and outlines the future directions of the research.

The main work presented in this thesis describes proteomics strategies applied to study the trafficking and turnover of glutathione reductase (Glr1) isoforms in the cytosol and mitochondria of Saccharomyces cerevisiae. Additional work was performed in order to understand mass spectrometric response factors and how they can affect peptides representation in the mass spectra. The opportunity to study two sub proteomes involved in biofilm formation of Pseudomonas aeruginosa PAO1 arose during my PhD and their analysis is also presented. Glr1 is a low abundant protein involved in the defence mechanisms against reactive oxygen species, which are sources of many diseases. Because of its biological relevance, considerable effort has been made in order to understand its functional role in the cell. This protein has been studied using biochemical strategies. In yeast, the cytosolic and mitochondrial forms of glutathione reductase are expressed by the same gene, GLR1, using alternative start codons. Translation from the first AUG codon generates the mitochondrial form incorporating a transit peptide necessary for import into the mitochondria. If the translation starts at the second AUG codon, the cytosolic counterpart is generated. Biochemical approaches show that the first AUG codon is not in favourable context and it has been suggested that leaky scanning accounts for the abundance of the cytosolic protein. The analysis of Glr1 forms by mass spectrometry was demanded because only the N-terminal region is informative about similarities and differences between cytosolic and mitochondrial forms. The protein is also of low abundance in both cytosol and mitochondrial compartments. A genetically modified strain, over-expressing this protein was, therefore, used throughout this work in order to analyse glutathione reductase in the mitochondria. This was not possible with the wild-type strain. Because the first AUG codon is now in context, the over-producing strain (MORF) yields both cytosolic and full length mitochondrial isoforms in the cytosol. Analysis of the mitochondrial protein shows that the cleavage of the pre-sequence is not specific. Three different forms of the mitochondrial N-terminal peptide were detected. Some attention was also devoted to glutathione reductase turnover in both cytosol and mitochondrial compartments using the genetically modified strain. Both N-terminal peptides generated from translation starting in the first and second AUG codon as well as mid-chain peptides from the cytosol fraction and one mid-chain peptide from the mitochondrial fraction, were used to calculate relative turnover measurements. My results illustrate that the mitochondrial protein is in faster turnover than the cytosolic counterpart. Moreover, the long and short forms observed in the cytosol also show slightly different turnover rates, the long form presenting faster turnover than the short form. Rapid turnover therefore maintains the level of glutathione reductase in the mitochondria. Despite the exquisite sensitivity of mass spectrometry, its restricted dynamic range compared with the dynamic range of the entire proteome is limiting for such studies. Peptides have different responses in the mass spectrometry and factors such as hydrophobicity and gas-phase basicity, can also contribute to low detectability of some peptides. To maximise the mass spectrometric response of peptides especially the ones derived from low abundant proteins, is extremely important. My thesis therefore includes a study of mass spectrometric response of peptides generated by different enzymes. Applying the kinetic method, the importance of the position of basic residues on gas-phase basicity and thus on the mass spectrometric response was demonstrated. In addition, the opportunity to carry out a related study on the proteome analysis of membrane vesicles and matrix within biofilms of Pseudomonas aeruginosa PAO1 has arisen and results of this study were presented in the final results chapter. It is the first time that two-dimensional chromatography was applied to analyse these sub-proteomes. Moreover, previous studies were mostly limited to the planktonic population; here the proteomes of membrane vesicles and extracellular matrix with the biofilm were addressed.

(2R)-N-Benzyl-2-acetamido-3-methoxypropionamide (lacosamide) is an anticonvulsant (Choi et al., 1996); under the brand name "Vimpat" this small molecule has recently been approved by the European Medicines Agency and the U.S. Food and Drug Administration for the treatment of epilepsy. The purpose of the research reported here is to develop and apply mass spectrometry approaches to the determination of protein targets of this novel therapeutic. The general strategy involves selecting potential target proteins using lacosamide analogues incorporating an 'affinity bait' to enable covalent modification binding to target proteins, and a 'chemical reporter' for the selective recovery of modified proteins. Lacosamide analogues are incubated with biological samples (primarily mouse brain extracts) and the modified proteins are recovered by introduction of a biotin tag (via the chemical reporter group). Streptavidin affinity chromatography is then used to enrich for bound molecules. The enriched proteins are subjected to tryptic digestion and the resultant peptides analysed by reversed phase liquid chromatography coupled with tandem MS, enabling recognition of proteins via database searching. Firstly, mass spectrometric characterisation of the biotinyl (R)-lacosamide analogue bound to model compounds was performed. Adducts with protected lysine, neurotensin and enolase were analysed. The data showed that ESI was more suitable for ionisation of modified peptides and proteins than MALDI. The biotin enrichment strategy was applied to mouse brain lysate to identify putative candidate target proteins. Twenty-eight candidate target proteins were identified. Moreover, the 14-3-3 protein family, CRMP2 and the sodium/potassium-transporting ATPase family showed preference for the biologically active(R)- isomer over the (S)- lacosamide analogue using a fluorescence tag. Three more biotinyl lacosamide analogues containing different affinity baits were used to enrich candidate target proteins of lacosamide. Most of the identified target proteins supported the findings of the analogue A. To indicate the binding sites, a method was developed for enriching peptides modified by the biotinyl (R)-lacosamide analogue, using streptavidin beads and subsequently analysed these biotinylated peptides using CID and ETD fragmentation methods. Neither fragmentation technique was optimal for elucidation of the sequence or site of modification of unknown target peptides. Purified recombinant proteins were therefore adducted with an AB-(R)-lacosamide analogue lacking the biotinprobe. This smaller (R)-lacosamide analogue underwent less fragmentation than the biotin analogue during CID and could be used for sequence and site identification of the modified peptides. In summary, the studies illustrated the power of MS to study drug mechanisms via the discovery of candidate protein targets.

No description available.

No description available.

The technique of electrothermal atomization atomic absorption spectrometry (ETA-AAS) is widely used for the analysis of samples originating from many sources, such as from the industry and from the environment. Many of these analyses which are performed, are vital for the operation, quality control and monitoring of the products produced in the industry, as well as the byproducts and effluents produced by these processes. Unfortunately, many of these analyses are prone to interferences, which lead to troublesome, inaccurate and costly analyses. Traditionally, chemical modifiers were used to overcome these interferences, but owing to certain limitations of these modifiers, a need arose for a new type of modifier. The envisaged modifier must be able to improve the quality of analyses by improving the sensitivity, detection limits and versatility of the conventional chemical modifiers. This investigation involves the use of a low pressure argon glow discharge. The discharge will sputter a solid metal chemical modifier, such as iridium or rhodium, onto the inside of a graphite tube to produce a permanent modifier. To begin with, the operational characteristics and optimization of the sputtering process for each of the modifiers were investigated. Secondly, the ashing curves and other analytical performance characteristics were determined for Pb and As in three matrices, namely sulphate, nitrate and chloride using the permanent modifiers. The analytical characteristics of the permanent modifiers were compared to the performances of the conventional chemical modifiers and comparisons and conclusions were drawn as to the performance and effectiveness of the permanent modifiers. / Dissertation (MSc (Chemistry))--University of Pretoria, 2007. / Chemistry / unrestricted

New measurements of the masses of the isotopes⁸,⁹,¹¹Li were made using recently commissioned TITAN Penning trap mass spectrometer at TRIUMF. The measurement of the halo nucleus ¹¹Li represents a new standard in Penning trap mass spectrometry, as it is the shortest lived, t₁/₂ = 8.8 ms, isotope ever weighed using this technique. Low energy, E = 20 keV, beams of these radioactive isotopes were produced using the ISAC facility. These were subsequentlycooled and bunched using a square-wave-driven Radio- Frequency Quadrupole (RFQ) ion guide, which was filled with hydrogen gas. The cooled ion bunches were then passed into a Penning trap where the mass measurements were made. A description of the RFQ in the ISAC hall is given along with some results from the commissioning of the device. A new set of harmonic deceleration optics is presented which have been successfully used to inject ions into the RFQ. Cooling of lithium ions with high DC efficiencies of 20%, in helium, and 40%, in hydrogen, are shown. Extraction of extremely short ion bunches, 30 ns FWHM, is also demonstrated. Storage times for stable lithium ions in helium and hydrogen were investigated. It was found that lithium ions could be cooled in hydrogen for up to 30 ms without significant losses whereas cooling in helium lead to exponential losses with a half-life of 5.7(1)ms. The TITAN Penning trap is described and the ⁸,⁹,¹¹Li data presented. Final values for the mass excess of ∆(⁸Li) = 20945.70(38) keV, ∆(⁹Li) = 24954.80(60) keV and ∆(¹¹Li) = 40728.1(12) keV are obtained. The ⁹,¹¹Li results are then used to obtain a new value for two neutron separation energy of ¹¹Li, S₂n = 369.3(1.3) keV. This agrees with the recent measurement from the MISTRAL spectrometer, 376(5) keV, at the two sigma level, but shows over three standard deviations from the most recent atomic mass evaluation, 300(20) keV / Science, Faculty of / Physics and Astronomy, Department of / Graduate

Ratio recording methods in mass spectrometry have hitherto been successfully used in analyses involving only a few of the lighter elements. In all these cases a great improvement in precision has resulted. This thesis describes a means whereby grid ratio recording can be extended to the precise measurement of complex spectra in the high mass range. In particular, lead isotope studies in geophysics provide a great and continuing need for increased analytical precision. The project has involved the construction of a complete mass spectrometer which incorporates two main features which have not been used before in mass spectrometry. The first of these is a scanning mechanism which moves the collector across the stationary ion beams. It is this innovation which has made possible the development of grid ratio recording. Since the total ion beam is stationary, variations in its intensity during the scanning process can be monitored accurately. A servo-divider system is arranged to make the measuring system Insensitive to variations in the total ion beam by recording the individual ion beam currents as fraction of the total Ion beam. The ratio system will effectively reduce large variations in the total Ion beam due to changes in source conditions by a factor of fifteen. Smaller variations are reduced to below the noise level existing in the recording system itself. The second principle which is believed to be new to mass spectrometry is the method by which the results of an analysis are presented by the instrument. An expanded scale system of recording has been developed whereby the amplitude of an ion current is presented as a digit on a counting tube, with the remainder displayed as a peak on a chart recorder. It is demonstrated that the expanded scale arrangement will record a known signal with a standard deviation of less than 0.02%. / Science, Faculty of / Physics and Astronomy, Department of / Graduate