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This dissertation details the design and implementation of a state-of-the-art ambient trace analysis technique known as laser electrospray mass spectrometry. This novel technique utilizes an intense, nonresonant femtosecond laser pulse to transfer nonvolatile, fragile molecules into the gas phase from various substrates. The vaporized analyte is subsequently captured, solvated and ionized in an electrospray plume enabling mass analysis. Laser electrospray mass spectrometry is capable of analyzing samples in the liquid or solid states, mass spectral imaging of adsorbed molecules and detecting low vapor pressure analytes remotely. Experiments with biomolecules and pharmaceuticals, such as vitamin B12 and oxycodone, have demonstrated that the nonresonant femtosecond laser pulse allows for coupling into and vaporization of all molecules. This implies that sample preparation (elution, mixing with matrix and choosing samples with a particular electronic or vibrational transition) is not necessary, thus creating a universal mass analysis technique. Investigations using low vapor pressure molecules, such as lipids and proteins, led to the discovery that unfragmented molecules are transferred into the gas phase via a nonthermal mechanism. The laser electrospray mass spectrometry technique has allowed for the nonresonant femtosecond laser vaporization and mass analysis of trace amounts of a nitro-based explosive from a metal surface. The vaporization of unfragmented explosive molecules from a surface facilitates the identification of the explosive, reducing the probability of false positives and false negatives. In addition, this "soft" vaporization of molecules using nonresonant femtosecond laser pulses allows for protein to be transferred from the condensed phase into the gas phase without altering the molecule's structure, enabling ex vivo conformational analysis and possible disease typing. / Chemistry

The use of femtosecond (fs) laser pulses in laser-induced breakdown spectroscopy (LIBS) and for chemical analysis using mass spectrometry is explored. A comparison of fs-LIBS and remote filament-induced breakdown spectroscopy (R-FIBS) in the analysis of graphite composites yielded more accurate results with filaments due to intensity clamping within the filament. The investigation of fs-LIBS and R-FIBS in the detection of explosives led to the discovery of femtosecond vaporization of intact molecules under ambient conditions. This knowledge was then used in the development of a new ambient laser-based mass analysis technique. The combination of nonresonant femtosecond laser vaporization with electrospray post-ionization called laser electrospray mass spectrometry (LEMS) was investigated as a universal detection method of pharmaceuticals, biological macromolecules and plant tissues. We show the capability of femtosecond lasers to desorb sample without any sample preparation or resonant transition in the sample or substrate. Ambient mass spectral imaging and tissue type classification is also demonstrated. / Chemistry

<P> Multi-site phosphorylation of protein targets by specific kinases is a common event used to propagate biological messages through signal transduction pathways in the context of the cellular environment and is a vital regulatory mechanism for many metabolic processes. Recent advances in the study of the protein glycogen synthase kinase-3 (GSK-3) have shed some light on the intricate role this enzyme plays within the framework ofmammalian cellular metabolism. Abnormal behaviour of GSK -3 profoundly impacts cellular function, and is implicated in Alzheimer's disease and the development of Type II Diabetes. A key issue in assaying the activity of GSK-3 is the ability to distinguish between singly and multiply phosphorylated substrates, as this enzyme has the ability to selectively phosphorylate a previously phosphorylated (primed) substrate. Given the serious nature of the disorders caused by the dysfunction of this kinase, high throughput screening of specific inhibitors from compound libraries is urgently needed. Unfortunately, many of the currently existing kinase screening technologies are geared towards monitoring single phosphorylation events and thus, are not be amenable to effective assaying of multiply phosphorylated substrates. In this thesis, a novel, solution-based assay method based on electrospray ionizationtandem mass spectrometry (ESI-MS/MS) is developed as a platform for inhibitor screening with full consideration being given to the specific nature of GSK-3 substrates and products. The semi-automated application of this assay is possible using an in-line autosampler, and is shown to be a potentially effective means for screening primed binding site inhibitors from compound mixtures, with subsequent deconvolution performed to isolate the effective molecule. Optimization of the MS-based assay required significant alterations in buffer conditions compared to those used in the standard GSK-3 radioassay based on y-32P ATP, owing to the inability of electrospray ionization to tolerate high buffer concentrations. Preliminary screening of mixtures was demonstrated, and expansion to screening of large compound libraries consisting of previously untested compounds and natural product extracts should be possible. </p> <p> To investigate the adaptation of the GSK-3 MS/MS assay to allow mixture deconvolution, a preliminary study was performed on the utilization of sol-gel technology for entrapment of GSK-3 to develop a solid-phase affinity assay for pull-down of bioactive ligands identified in enzyme activity assays. This method requires the preservation of enzyme function within the silica matrix, which has not been previously demonstrated for GSK-3. The sol-gel entrapment of GSK-3, however, proved to be problematic. Implementation of a flow-through assay using immobilized GSK-3 was hampered by issues such as non-specific adsorption of the cationic substrate and inhibitors, owing to electrostatic interactions with the anionic silica matrix used for enzyme entrapment. Future work aimed at further developing and optimizing the sol-gel materials and processing methods are proposed. </p> / Thesis / Master of Science (MSc)

<p> 0-GlcNAc modification is a single N-acetylglucosamine (GlcNAc) modification on Ser or Thr residue on protein. The addition and removal of the 0GlcNAc molecule are controlled by two enzymes (OGT and NCOAT). In this study, I expressed and purified the two enzymes involved in the 0-GlcNAc modification. A method was developed for the synthesis and purification of the peptide substrate YSDSPSTST for in vitro glycosylation and characterized the OGT enzyme activity by the in vitro glycosylation and H3 labeling. A method was developed based on detection of glycosylation peptide by mass spectrometry after separation by capillary liquid chromatography (CapLC). The optimization of mass spectrometry parameters was done using synthesized standard glycopeptide YSDSPSgTST ("Sg" represents 0-GlcNAc modified Serine). The in vitro modification site was determined by CID after alkaline J3-elimination. Furthers experiment could include detection of 0-GlcNAc modification of protein substrate both in vitro and in vivo. This will give a better understanding of the dynamics of 0-GlcNAc modification. </p> / Thesis / Master of Science (MSc)

Metabolomics is the systematic analysis of low-molecular weight compounds (metabolites) within biological systems that represent molecular endpoints of gene expression and environmental exposures. A major goal of metabolomics is achieving better understanding of the pathophysiology of complex disease processes while elucidating mechanisms of action of nutrients, toxins, and/or drugs. Multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) is a high-throughput microseparation platform that is ideal for the analysis of polar/ionic metabolites from volume-restricted biological samples. This thesis includes two major metabolomics projects using MSI-CE-MS that are aimed at contributing new advances in public health and chronic disease prevention. Chapter II presents an analysis of the metabolome from patients with phenylketonuria (PKU) — a genetic disease affecting phenylalanine (Phe) metabolism that requires lifelong dietary restriction to prevent irreversible intellectual disabilities. A targeted and nontargeted metabolomics approach using matching urine and plasma samples was conducted to confirm known markers of PKU and identify new markers associated with dietary adherence and disease progression. Along with increased excretion of Phe catabolites in urine, high plasma Phe was associated with decreased excretion of acylcarnitines and greater excretion of histidine catabolites, suggesting impaired fatty acid oxidation and micronutrient deficiencies, respectively. Overall, this may provide a strategy to objectively monitor dietary adherence beyond standard dietary records or patient recall. Chapter III investigates the impact of electronic cigarette smoke exposure on the placental metabolome as a model cell line of fetal development. Evidence of altered amino acid metabolism, in addition to changes in acylcarnitines and metabolites associated with cellular proliferation, were observed in more susceptible first trimester placental cells and were attributed to flavouring agents irrespective of nicotine dosage. This work supports the hypothesis that flavoured e-cigarette formulations pose a significant health risk in comparison to unflavoured formulations and supports the need for further risk assessment and careful regulation of these products to prevent deleterious birth outcomes in pregnant mothers. / Thesis / Master of Science (MSc)

The introduction of ionization under ambient conditions has greatly simplified mass spectrometric analysis. Over past decade, ambient ionization mass spectrometry (MS) methods have revolutionized the way complex samples are analyzed under environmental conditions without requiring, in most cases, any sample pretreatment. Ambient ionization MS gained popularity among other analytical techniques due to its simplicity and its suitability for analysis of small and large molecules. However, ambient ionization methods can suffer from low accuracy and sensitivity due to matrix effects and interferences within complex samples, as well as from poor ionization efficiency. Matrix effects in ambient ionization are usually caused by ion suppression and may depend on different factors, e.g. matrix-to-analyte concentration ratios, proton affinities of analyte and matrix species. To overcome these challenges, in this thesis we present a new approach where a probe is used both as a direct sampling device and as an efficient ambient ionization source. This approach leverages high surface area gold electrodes, fabricated through low-cost bench-top fabrication methods and functionalized using self-assembled alkyl thiol monolayers, as functional conductive sampling probes (FCSPs) for the extraction and concentration of analytes from a sample solution. FCSPs loaded with the targeted analytes were then used to demonstrate a new and highly efficient ionization approach, called Primary Ion Mass Spectrometry Source (PIMSS). In this approach, following capture, the bound analytes are directly desorbed into the mass spectrometer, where ionization is achieved solely through the extraction voltage applied to the probe. 3D-printing was used to design an interface to couple FCSPs to the mass spectrometer. In this work, we discuss a detailed method development and optimization stage and present capabilities of the proposed assay. / Thesis / Master of Science (MSc)

Oxidative stress, described as the state of disturbed intracellular redox balance, has been associated with several human conditions including ageing, apoptosis, cancer, autoimmune and neuro-degenerative diseases. Stress studies have shown that reactive oxygen species (ROS) and reactive nitrogen species (RNS) along with its intermediates can attack essential cell targets such as: DNA, proteins, lipids and carbohydrates, leaving behind dysfunctional biologic molecules. In effect, a cell's primary response is to involve several defense mechanisms that are under a complex and intricate regulatory control to repair any damages that may have occurred. Although several stress studies have been conducted in the past that have approached this biologically complex process step by step, application of a Systems Biology towards a comprehensive understanding is still emerging. The current objective of this project is to identify proteins that change in response to cumene hydroperxoide (CHP) treatment and in parallel make an attempt to uncover events and processes that are a part of CHP-induced oxidative stress response. From a systems biology viewpoint, the Yeast Oxidative Stress project will monitor response at three different levels: transcriptomics, proteomics and metabolomics, with dynamic changes being measured from 3 to 120 min after CHP addition. Data collected from the different levels will be integrated to accomplish a holistic viewpoint of stress response in the given system and to develop mathematical tools for modeling biochemical networks. Saccharomyces cerevisiae was chosen as a model, based on its availability of a completely mapped genome sequence with a collection of null mutants that was relevant to our fundamental research of stress response mechanism. Yeast, a simple unicellular eukaryote has been extensively used for applied studies and has proven to be indispensable for stress research. Information derived from this project can reveal response mechanisms used by higher eukaryotes, especially if via analogous signaling cascades that are comparable between organisms. Current research investigates an optimal workflow for generating 2D gel based protein expression data and identifying proteins that are induced by cumene hydroperoxide treatment. A non-targeted protein profiling followed by a 2-way ANOVA analysis provided a list of proteins that differ significantly between treatments. Protein identification provided relevant information on which proteins are affected by CHP induced stress response, including posttranslational modifications of peroxiredoxins. Redox active protein, Ahp1, was regulated post-translationally with sulfonic acid modification observed for its active Cys(62) residue. / Ph. D.