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Ip Wai-Ho Herman. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 87-90). / Abstracts in English and Chinese. / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iii / Acknowledgement --- p.iv / Declaration --- p.v / Table of Content --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Fourier transform ion cyclotron resonance mass spectrometry (FRICR) --- p.2 / Chapter 1.1.1 --- History of FTICR --- p.2 / Chapter 1.1.2 --- Theory of FTICR --- p.4 / Chapter 1.1.3 --- FTICR with electrospray ionization source --- p.10 / Chapter 1.2 --- Tandem mass spectrometry --- p.11 / Chapter 1.2.1 --- Introduction --- p.11 / Chapter 1.2.2 --- Collision-induced dissociation --- p.12 / Chapter 1.2.3 --- Surface-induced dissociation --- p.12 / Chapter 1.2.4 --- Photodissociation --- p.13 / Chapter 1.2.5 --- Blackbody infrared radiative dissociation (BIRD) --- p.13 / Chapter 1.3 --- Electron capture dissociation --- p.13 / Chapter 1.4 --- Recent advances in ECD experiments --- p.15 / Chapter 1.5 --- Outline the present work --- p.17 / Chapter 2. --- EXPERIMENTAL AND INSTRUMENTATION --- p.18 / Chapter 2.1 --- Instrumentation --- p.19 / Chapter 2.1.1 --- Fourier-transform ion cyclotron resonance mass spectrometer --- p.19 / Chapter 2.1.2 --- Vacuum system --- p.19 / Chapter 2.1.3 --- Electrospray ionization source --- p.23 / Chapter 2.1.4 --- Electrostatic ion focusing system --- p.26 / Chapter 2.1.5 --- Infinity cell --- p.28 / Chapter 2.1.6 --- Electron emission source --- p.29 / Chapter 2.1.7 --- Data acquisition system --- p.32 / Chapter 2.2 --- Experimental --- p.32 / Chapter 2.2.1 --- Simple acquisition pulse program --- p.32 / Chapter 2.2.2 --- ECD pulse program with/without collision cooling --- p.35 / Chapter 3. --- OPTIMIZATION OF EXPERIMENTAL PARAMETERS FOR ELECTRON CAPTURE DISSOCIATION --- p.38 / Chapter 3.1 --- Introduction --- p.39 / Chapter 3.2 --- Experimental --- p.40 / Chapter 3.2.1 --- Materials --- p.40 / Chapter 3.2.2 --- Sample preparation --- p.41 / Chapter 3.2.3 --- Instrumentation --- p.41 / Chapter 3.3 --- Results and discussion --- p.42 / Chapter 3.3.1 --- Benchmark conditions --- p.42 / Chapter 3.3.2 --- Effect of the filament heating currents (If) --- p.45 / Chapter 3.3.3 --- Effect of the average filament bias voltages (Vf) --- p.49 / Chapter 3.3.4 --- Effect of the electron irradiation time (te) --- p.53 / Chapter 3.3.5 --- Reduction of the fragment ions intensity --- p.56 / Chapter 3.3.6 --- Effect of the trapping potentials --- p.60 / Chapter 3.4 --- Conclusions --- p.62 / Chapter 4. --- ENHANCEMENT ON ELECTRON CAPTURE DISSOCIATION EFFICIENCY --- p.63 / Chapter 4.1 --- Introduction --- p.64 / Chapter 4.2 --- Experimental --- p.65 / Chapter 4.2.1 --- Materials --- p.65 / Chapter 4.2.2 --- Sample preparation --- p.65 / Chapter 4.2.3 --- Instrumentation --- p.65 / Chapter 4.3 --- Results and discussion --- p.66 / Chapter 4.3.1 --- Effect of the filament position --- p.66 / Chapter 4.3.2 --- Effect of the collision gas pressure --- p.68 / Chapter 4.3.3 --- Effect of the collision gas --- p.73 / Chapter 4.3.4 --- Effect of the electron irradiation at different pulse gas interval --- p.75 / Chapter 4.3.5 --- Effect of the multiple electron irradiation --- p.76 / Chapter 4.3.6 --- Optimized Conditions --- p.79 / Chapter 4.4 --- Conclusions --- p.84 / Chapter 5. --- CONCLUSIONS --- p.85 / Chapter 5.1 --- Conclusions --- p.86 / REFERENCES --- p.87 / APPENDIX --- p.91 / Appendix A Simple pulse sequence program for ESI FTICR-MS experiments --- p.91 / Appendix B Pulse sequence program for ESI FTICR-MS electron capture dissociation --- p.95 / Appendix C Pulse sequence program for ESI FTICR-MS electron capture dissociation experiments with collision cooling --- p.99 / Appendix D Modified pulse sequence program for ESI FTICR-MS electron capture dissociation experiments with a time lag between collision cooling and electron irradiation. --- p.104 / Appendix E Modified pulse sequence program for ESI FTICR-MS electron capture dissociation experiments with multiple irradiation --- p.109

Redox potential, a measure of how oxidising or reducing an environment is, is tightly regulated by cells to minimise detrimental chemical oxidation and reduction reactions. In proteins, it is the sulfur containing cysteine residues that can be post-translationally modified through specific redox reactions, for example, the formation of disulfide bonds between cysteine residues can be crucial to protein structure. It has recently been hypothesised that signalling pathways utilising redox regulated proteins may be arranged into electrochemical series. The characterisation of the redox properties of specific cysteine residues in proteins has proven difficult using traditional redox characterisation methods such as cyclic voltammetry. A number of biochemical methods have been developed for studying the effect of the redox environment on proteins, many making use of mass spectrometry and allowing for localisation of the site of the modification to specific cysteine residues. However, fewer methods have been reported that facilitate accurate quantification for the determination of the mid-point potential of these redox regulated cysteine residues. Here, a differential labelling protocol using high resolution mass spectrometry techniques for the study of redox chemistry of cysteine residues in proteins will be reported. The protocol exploits the novel chemistry of thiol groups for specific alkylation and allows for both qualitative and quantitative experiments. Thioredoxin-1 from E. coli and human systems was used as a model protein and a novel disulfide bond was characterised. The reducing potential of the active site cysteine residues of human thioredoxin were found to be very similar to those of the E. coli proteoform, -276 ± 1 and -281.4 ± 0.3 mV respectively. The remaining three cysteine residues of human thioredoxin were found to be regulated at more oxidising potentials. The protocol developed was applied to a protein from the cell death pathway of apoptosis; human caspase-3 is an executioner protease from the caspase cascade. Caspase-3 was found to contain three redox sensitive cysteine residues. The catalytically active cysteine residue was redox regulated via two mechanisms, glutathionylation and disulfide bond formation. One of these mechanisms gives the active site cysteine residue a calculated reducing potential of -165 ± 6 mV supporting the correlation between caspase-3 activity and its observed role in the apoptotic pathway but not in necrotic cell death.

No description available.

Glycoproteins, and their subset proteoglycans, are an important group of molecules in joint tissues, providing crucial functions such as cartilage structural integrity and lubrication at cartilage surfaces. The functionality of these glycoproteins is attributable to their oligosaccharide components, however surprisingly little is known about their fine structural details. With the use of glycoproteomic methods, this thesis presents the development and incorporation of mass spectrometric, biochemical and immunological methods to elucidate glycoprotein structures in synovial fluids, chondrocytes and synoviocytes in order to provide insight into how their structures may contribute to their functions. Initially, anion exchange chromatography was used to extract the acidic fraction containing glycoproteins and proteoglycans in arthritic synovial fluid (SF) samples, followed by proteomic analysis to identify the main glycoproteins in 1D-SDS-PAGE gels. To complement these findings, an in-gel enzymatic digest method for glycosaminoglycan (GAG) and oligosaccharide analysis was developed for analysis of glycoproteins by graphitised carbon liquid chromatography mass spectrometry (LC-MS). Further characterization of the major glycoprotein, lubricin, was pursued by investigating its interactions with the surrounding extracellular matrix (ECM) from its cellular sources and characterising the secreted lubricin with Western blot and proteomic analysis. Finally, the graphitised carbon LC-MS method was applied to analyse the overall glycosylation profiles of lubricin. The major glycoprotein found in arthritic synovial fluid was lubricin, as identified by peptide LC-MS and Western blot. Graphitised carbon LC-MS identified the major chondroitin sulfate (CS) repeat region disaccharides and linkage region oligosaccharides of aggrecan with confirmation through tandem mass spectra and Western blots using CS linkage region stub antibodies. Application of this method to lubricin led to the discovery of O-linked oligosaccharide structures which were previously undescribed for lubricin. A higher proportion of sialylated oligosaccharide structures were detected in the rheumatoid arthritis (RA) samples compared to the osteoarthritic (OA) samples, which signifies a diagnostic difference between these diseases. Sulfated oligosaccharide structures were also detected on synovial fluid lubricin, correlated with Western blot reactivity with the MECA-79 antibody, thus suggesting a role for lubricin in inflammation. Overall the results demonstrated that glycosylation structure indicates additional functional properties for the glycoproteins such as lubricin.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2009 Aug 16

Thesis (Ph. D.)--Oregon Graduate Center, 1979.

A single-ultrafine-particle mass spectrometer was constructed and deployed for size-resolved ultrafine aerosol composition measurements during the winter of 2002-2003 in College Station, Texas. Three separate experiments were held between December and March with six week intervals. Almost 128,000 mass spectra, corresponding to particles with aerodynamic diameters between 35 and 300 nm, were collected and classified. Fifteen statistically significant classes were identified and are discussed in this paper. Nitrate, potassium, carbon, and silicon/silicon oxide were the most frequently observed ions. Nitrate was present in most of the particles, probably due to the agricultural activity in the vicinity of the sampling site. The nitrate detection frequency was found to be sensitive to the ambient temperature and relative humidity. Another particle class, identified as an amine, exhibited strong relative humidity dependence, appearing only during periods of low relative humidity. There is evidence that some of the detected particles originated from the large urban centers, and were coated with nitrate, sulfate, and organics during transport.

In this work, the radiolysis mechanisms in solids as well as in liquid and frozen aqueous solutions have been studied. Liquid chromatography tandem mass spectrometry has evolved so that mass spectral information can now be used to determine the most probable structures of radiolysis products, even those present in traces amounts. Theoretical routes explaining the formation of radiolysis products can be deduced from their structures. The development of strategies to limit the degradation of the active pharmaceutical ingredient during irradiation of the drug requires a better knowledge of the radiolysis mechanisms responsible for the drug degradation. Metoclopramide, an antiemetic drug, has been selected as a model, due to the variety of its chemical bonds. In the solid state, radiation-induced degradation of the drug was very low (<0.1%) and only four radiolysis products were detected in traces. The “major” radiolysis product was formed after the loss of the chlorine by dissociative electron capture. For metoclopramide liquid aqueous solutions, the loss of the drug was important (~30% loss at 25 kGy) and several radiolysis products were detected. The majority of the degradation products were generated following the attacks of hydroxyl radicals and aqueous electrons. The loss of metoclopramide could be lowered up to acceptable levels (<10% loss) provided that radioprotective additives were added and the irradiation dose was limited to 15 kGy, which could be sufficient to reach the required SAL. The selected excipients were mannitol (which reacts mainly with the hydroxyl radical), nicotinamide and pyridoxine that react with both the aqueous electron and the hydroxyl radical. The irradiation of frozen aqueous solutions allowed to minimize the loss of active substance even for a 25 kGy dose. This approach seems to be the most promising method for terminal sterilization of aqueous solutions by ionizing radiations. The major radiolysis product was formed after the attack of the electron. Some of the radiolysis products detected were attributed to the attack of the hydroxyl radical, demonstrating the feasibility of a reaction between the hydroxyl radical from ice radiolysis and the solute. A comparison was performed with irradiated frozen solutions of metoprolol, which has been studied in liquid aqueous solutions (C. Slegers’ thesis). Degradation of metoprolol when irradiated in frozen solutions was negligible.