No description available.

Mass spectrometry has been recognized as a prominent analytical technique for peptide and protein identification and quantitation. With the advent of soft ionization methods, such as electrospray ionization and matrix assisted laser desorption/ionization, mass spectrometry has opened a new era for protein and proteome analysis. Due to its high-throughput and high-resolution character, along with the development of powerful data analysis software tools, mass spectrometry has become the most popular method for quantitative proteomics. Stable isotope labeling and label-free quantitation methods are widely used in quantitative mass spectrometry experiments. Proteins with stable expression level and key roles in basic cellular functions such as actin, tubulin and glyceraldehyde-3-phosphate dehydrogenase, are frequently utilized as internal controls in biological experiments. However, recent studies have shown that the expression level of such commonly used housekeeping proteins is dependent on cell type, cell cycle or disease status, and that it can change as a result of a biochemical stimulation. Such phenomena can, therefore, substantially compromise the use of these proteins for data validation. In this work, we propose a novel set of proteins for quantitative mass spectrometry that can be used either for data normalization or validation purposes. The protein set was generated from cell cycle experiments performed with MCF-7, an estrogen receptor positive breast cancer cell line, and MCF-10A, a non-tumorigenic immortalized breast cell line. The protein set was selected from a list of 3700 proteins identified in the different cellular sub-fractions and cell cycle stages of MCF-7/MCF-10A cells, based on the stability of spectral count data (CV<30 %) generated with an LTQ ion trap mass spectrometer. A total of 34 proteins qualified as endogenous standards for the nuclear, and 75 for the cytoplasmic cell fractions, respectively. The validation of these proteins was performed with a complementary, Her2+, SKBR-3 cell line. Based on the outcome of these experiments, it is anticipated that the proposed protein set will find applicability for data normalization/validation in a broader range of mechanistic biological studies that involve the use of cell lines. / Master of Science

Naphthenic acids (NA) are a highly complex mixture of aliphatic carboxylic acids that may contain multiple rings and unsaturated double bonds, and are a subset of the naphthenic acid fraction components (NAFC), which can contain heteroatoms, unsaturations, and aromatic structures. Mono-carboxylated NAs can be classically represented by CnH2n+zO2 where z is a negative integer representing the hydrogen deficiency. NAs and NAFCs are components of the acid extractable organics (AEO) frequently associated with increased toxicity and observed at elevated concentrations in oil sands process waters (OSPW). Numerous chromatographic and mass spectrometry techniques have recently emerged to probe the composition and concentrations of these components. This thesis reports the use of a capillary hollow fiber polydimethylsiloxane (PDMS) membrane mounted on a probe interface that can be immersed directly into an aqueous sample. A methanol acceptor phase passing through the lumen transports analyte to an electrospray ionization source and a triple quadrupole mass spectrometer. This technique, termed condensed phase membrane introduction mass spectrometry (CP-MIMS), allows for rapid screening of m/z profiles and on-line quantification of NAs in complex samples within minutes. This thesis reports parametric studies of several model carboxylic acids and a standard naphthenic acid mixture (Merichem) involving the effect of sample pH on membrane transport and acceptor phase pH on ionization enhancement. Several quantitative strategies are explored including the use of an internal standard in the acceptor phase to correct for ionization suppression and variations in instrument sensitivity, and the use of selected ion monitoring (SIM) experiments to increase analytical sensitivity and potentially target specific NA isomer classes for quantitation. Analytical performance measures such as the linear dynamic range (1-2300 ppb [NA]T as Merichem), sensitivity (~1 ppb [NA]T as Merichem detection limit), precision (~20 %RSD for replicates of a single OSPW) and accuracy are reported. Quantitative results for various OSPW samples in the ppb to ppm range are reported as equivalents of several surrogates, including 1-pyrenebutyric acid (PyBA), Merichem, and a large-volume extract of northern Alberta OSPWs. The variety of quantitation strategies allows results to be compared with several other published methods. CP-MIMS results for three mid-range northern Alberta OSPWs are compared to analysis by Environment Canada with an average -21% bias. Results for five archived OSPWs spanning a wider concentration are compared to data from AXYS Analytical, with an average -49% bias. Applications of CP-MIMS as an in-situ monitor of removal efficiencies of NAs on adsorbents and real-time mass profile changes are also presented, along with some interpretation of the resulting high-resolution kinetic data to obtain decay constants. Using the targeted SIM method, adsorption decay can be followed in real-time for various isomer classes within the Merichem mixture, and kinetic data extracted to obtain decay constants for each. CP-MIMS is also used to characterize adsorption behavior for two activated biochars, including % removals for various loadings of each when added to stirred Merichem solutions. Preliminary multi-loading experiments are conducted with one biochar, and the ability of CP-MIMS to characterize adsorbent behavior by constructing adsorption isotherm plots is demonstrated. / Graduate

In the last decade, dynamic combinatorial mass spectrometry (DCMS) with protein targets has emerged as a promising method for the identification of enzyme-inhibitors. 2-Oxoglutarate (2OG) oxygenases are involved in important biological processes related to many diseases; several human 2OG oxygenases are targeted for pharmaceutical intervention. This thesis describes inhibition studies on three 2OG oxygenases using DCMS and structure activity relation (SAR) studies. Disulphide based DCMS was used for the identification of N-oxalyl based lead inhibitors for the 2OG oxygenase AlkB from Escherichia coli. Crystallographic analyses of AlkB with a lead inhibitor assisted in the design of a second generation of inhibitors using N-oxalyl, pyridyl and quinolinyl scaffolds. Crystallographic and kinetic data of three potent and selective AlkB inhibitors validates the DCMS approach for the development of 2OG oxygenase inhibitors. The hypoxia inducible factor hydroxylase, prolyl hydroxylase domain 2 (PHD2), was then used as the model enzyme for the development of a novel DCMS approach employing the reversible reaction of boronic acids with diols to form boronate esters. The ‘boronate’ DCMS method was used to identify pyridyl- substituted lead compounds. Further modification of the pyridine scaffold, based on structural analyses, led to the development of highly potent and selective PHD2 inhibitors. To identify inhibitors for the fat mass and obesity associated protein (FTO), another 2OG oxygenase, an inhibition assay was developed. The inhibition assay was used in conjunction with a differential scanning fluorimetry (DSF) binding assay to identify isoquinolinyl and pyridyl inhibitor scaffolds, related to those used in the DCMS studies. FTO complexed structures of these compounds, and with a natural product anthraquinone, enabled the design and synthesis of new inhibitors that are both co-substrate and substrate competitors of FTO. One such compound proved to be a potent FTO inhibitor with improved selectivity over other 2OG oxygenases. Overall, the work validates the use of DCMS methods for the development of potent and selective inhibitors for 2OG oxygenases, and by implication of other enzyme families.

One of the most fundamental challenges in analytical mass spectrometry (MS) is the efficient conversion of neutral molecules into intact gas-phase ions. In this thesis, I investigate the capabilities of various new and established ionization techniques including (a) the Array of Micromachined UltraSonic Electrosprays (AMUSE), (b) Direct Analysis in Real Time (DART) and (c) Electrospray Ionization (ESI) for bioanalytical and biomedical analysis purposes. The AMUSE is a MicroElectroMechanical System (MEMS)-based device that was created as an alternative, and more sensitive approach for ion generation in an array format. In the AMUSE, the processes of droplet formation and DC droplet charging are separated allowing ionization of liquid samples using low charging voltages and a wide variety of solvents. Our analytical characterization work with the AMUSE showed that ion generation with this device was indeed possible, and that incorporation of a Venturi device increased signal stability and sensitivity due to enhanced droplet desolvation and increased ion transfer efficiency. A detailed investigation to determine the optimal source parameters for ionization of aqueous solutions of model compounds including reserpine, leucine enkephalin and cytochrome C was carried out and it was found that ionization was possible even without the application of a DC charging potential. Subsequent experiments using the thermometer ion method to characterize the AMUSE from a more fundamental point of view, showed that AMUSE ions are lower in internal energy than ESI ions, opening interesting possibilities for the mass spectrometric study of labile species. Furthermore, it was found that it was possible to manipulate the internal energy of the ion population by varying the parameters that most strongly affect desolvation and focusing. Our studies with DART were directed at investigating its analytical potential for application to the identification of active ingredients (AIs) in low quality combination medicines and counterfeit antimalarials that are commonly sold in regions of the world (particularly Southeast Asia) where drug resistant malaria is endemic as their use may engender increased resistance against the few remaining effective antimalarials.

The novel instrumentation of nanomanipulation coupled to nanospray mass spectrometry and its applications are presented. The nanomanipulator has the resolution of 10nm step sizes allowing for specific fine movement used to probe and characterize objects of interest. Nanospray mass spectrometry only needs a minimum sample volume of 300nl and a minimum sample size of 300attograms to analyze an analyte making it the ideal instrument to couple to nanomanipulation. The nanomanipulator is mounted to an inverted microscope and consists of 4 nano-positioners; these nano-positioners hold end-effectors and other tools used for manipulation. This original coupling has been used to enhance the current abilities of cellular probing and trace fiber analysis. Experiments have been performed to demonstrate the functionality of this instrument and its capabilities. Histidine and caffeine have been sampled directly from single fibers and analyzed. Lipid bodies from cotton seeds have been sampled indirectly and analyzed. The few applications demonstrated are only the beginning of nanomanipulation coupled to nanospray mass spectrometry and the possible applications are numerous especially with the ability to design and fabricate new end-effectors with unique abilities. Future study will be done to further the applications in direct cellular probing including toxicology studies and organelle analysis of single cells. Further studies will be directed in forensic applications of this instrument including gunshot residue sampled from fibers.

No description available.

Digitized by Kansas Correctional Industries

Call number: LD2668 .T4 1956 H38 / Master of Science

Call number: LD2668 .T4 1953 C69 / Master of Science