Thesis (M.S.)--Brigham Young University. Dept. of Chemistry and Biochemistry, 2006. / Includes bibliographical references (p. 85-91).

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

Thesis (Ph. D.)--Oregon Graduate Center, 1988.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 144-158).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 145-152).

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references. Also available on the World Wide Web.

In this thesis, a miniaturised field asymmetric waveform ion mobility spectrometry (FAIMS) device is combined with mass spectrometry (MS), and liquid chromatography, for the development and application of bioanalytical methodologies. FAIMS is a highly orthogonal to MS and LC and has the potential to enhance both targeted and non-targeted bioanalytical applications. Chapter two demonstrates the capability of the FAIMS combined with mass spectrometry to reduce the complexity of the mass spectrum by separating species of different charge states and overlapping mass-to-charge ratios that are challenging to separate by MS. FAIMS selected transmission shows improvement in signal-to-noise ratios for low intensity species and enables visualisation of species undetectable without FAIMS. Chapter three describes the development of an LC-FAIMS-MS method for the rapid analysis of saliva for the identification of potential biomarkers as a result of oxidative stress. The combination of FAIMS showed a reduction in saliva matrix interferences resulting in improved discrimination and peak integration of two salivary oxypurine compounds in a rapid LC-FAIMS-MS method. Chapter four investigates the FAIMS separation of seven steroid metabolites with a range of cationic adducts, in order to develop a rapid screening LC-FAIMS-MS method for the determination of isobaric steroid metabolites in urine. LC-FAIMS-MS analysis of the steroid metabolites shows improved discrimination of co-eluting and isobaric steroid metabolites with improvements in signal-to-noise ratio with reductions in chemical noise, demonstrating the potential of combining FAIMS with LC-MS. Chapter five demonstrates the potential of FAIMS to increase peak capacity in non-targeted omics applications, by combining rapid compensation field scanning of the FAIMS with ultra-high performance LC-MS. The rapid scanning of the FAIMS allows acquisition of full scan FAIMS and MS nested data sets within the timescale of a UHPLC chromatographic peak, and is applied to the non-targeted profiling of human urine. Improvements in the number of features detected using LC-FAIMS-MS were as a result of reductions in chemical noise and separation of co-eluting isobaric species across the whole analytical space, demonstrating the potential of combining FAIMS with LC and MS.

Reversible protein phosphorylation, the best studied post-translational modification, regulates most cellular processes, including signaling, migration, cell cycle progression, DNA damage repair, stress response and modulaton of the activities of metabolic enzymes. Therefore, it has emerged as a key therapeutic target in diseases in which these processes are deregulated. Unlike kinases, protein phosphatase 1 (PP1) is a promiscuous enzyme that gains its substrate specificity from a large group of “regulatory subunits” with which it associates to form a range of holoenzyme complexes targeted to specific subcellular localizations and substrates. Inhibition of a specific dephosphorylation event therefore relies on targeting the regulatory rather than the catalytic subunit. The present study uses GFP as a molecular reporter to assess the localization of PP1c and identify the underlying binding events that govern it via a combination of fluorescence imaging, cellular fractionation, affinity purification and quantitative mass spectrometry. While there is some overlap in their targeting and intracellular roles, the three PP1 isoforms show distinct localizations based on relative preferences for particular regulatory subunits. In this study we assembled a comprehensive map of isoform- and compartment- specific phosphatase complexes in three different cultured human cell lines, using the data to extrapolate, with confidence, the distribution of each PP1 isoform between a large pool of known/predicted and novel regulatory subunits. Network analysis also highlighted key multiprotein complexes to which PP1 is targeted by these regulatory subunits, and identified a novel regulatory subunit that links phosphatase activity to regulation of protein degradation. Our work confirmed that Mypt1, the regulatory subunit that targets PP1 activity to the myosin light chain, preferentially associates with the beta isoform of PP1c. We further demonstrated that they are in complex in both the cytoplasm and nucleus, and represent ~30% of the total PP1β holoenzyme complexes in both interaphase and mitotic cells. Further investigation of these complexes led to the discovery of Specc1 and Specc1L, which associate with Mypt1/PP1β via direct binding to Mypt1. Specc1/1L are microtubule binding proteins that can also associate with actin filaments, and we demonstrated that they mediate the distribution of Mypt1/PP1β complexes between these two cytoskeletal networks. Given that disruption of this balance has been implicated in disease states including cancer and hypertension, the Specc1/L family represents a novel therapeutic target for the regulation of Mypt1/PP1 activity. With PP1 now emerging as a promising therapeutic target and the first PP1-targeted therapeutic drug, Sephin 1, in clinical trials, a better understanding of PP1’s in vivo distribution between holoenzyme complexes is essential. Our work establishes an initial “snapshot” of this distribution against which changes can be assessed, as we demonstrated here by showing its re-distribution in mitotic cells. Dynamic redistributions during specific cell processes such as differentiation or in response to perturbations or disease states can be assessed in a similar fashion in future, facilitating both identification of the relevant complexes and the design of specific strategies to target them therapeutically.

Cis-diol-containing metabolites have attracted increasing attention in recent years. These metabolites widely exist in the body fluids and tissues. They play important roles in the structure, function and metabolic activity of cells. Some of them are related to cell proliferation and metabolic processes. And they have been used to denote a state of disease as potential biomarkers. Several methods have been developed for the analysis of cis-diol-containing metabolites. However, these methods faced a challenge to separate and detect isomers of these compounds, particularly for compounds with low abundance and high polarity. Therefore, novel methods were necessary to improve the separation and detection sensitivity of this kind of metabolites. With this aim, chemical derivatization methods were developed for cis-diol-containing metabolite detection by using liquid chromatography-mass spectrometry in this project. These methods were optimized and validated to achieve the optimal reaction conditions. And they were applied to study real-world biological systems, including the changes of modified nucleosides in hepatocellular carcinoma (HCC) nude mice and toxic effects of bisphenol A (BPA) exposure. Firstly, the derivatization reaction of cis-diol compounds with acetone were optimized. Factors that affected reaction efficiency were investigated by reacting guanosine (G) with acetone. The optimal reaction conditions were validated by detecting four acetonides of urinary nucleosides by using LC-MS/MS. The results showed that the approach had good linearity, accuracy and precision. The recoveries were ranged from 92.9% to 103.5%. It indicated that the assay was reproducible. The robust method should be potentially useful for the analysis of modified nucleosides and other cis-diol-containing metabolites in biological samples. The validated derivatization method was applied to determine urinary nucleosides by LC-MS. This method not only improved the retention of nucleosides on reversed-phase column, but also reduced the matrix effect from urine samples and enhanced detection sensitivity of mass spectrometry. Isotope labeling method with acetone-d6 and multivariate statistical analysis enabled the positive identification of 56 nucleosides, including 52 modified nucleosides. The obtained results indicated that the derivatization method was practical, fast and effective for the identification of urinary nucleosides. It was successfully applied to study the changes of urinary nucleosides in nude mice bearing HCC. Some significantly changed nucleosides were identified as potential biomarkers. Subsequently, this approach was modified by employing parallel reaction monitoring (PRM) method which was based on high resolution MS to detect urinary nucleosides in rats exposed to BPA. Comparing to the data acquired by triple quadrupole MS with neutral loss scanning, higher specificity and sensitivity were achieved by using PRM scanning mode. Therefore, more nucleosides were identified by using the method in urine samples (from 56 up to 66). The changes of the detected nucleosides were studied in the rats exposed to BPA. Various trends of modified nucleosides were observed with different dose BPA exposure. Specifically, the high-dose exposure group was the most strongly affected. The biomarker of RNA oxidation, 8-hydroxyguanosine (8-oxoG), showed significant change in this group. It proved that BPA exposure could induce RNA damage when the dose of BPA was beyond a certain amount. Except for nucleosides, other cis-diol-containing metabolites, such as carbohydrates, were also studied by using the derivatization method. Acetone and acetone-d6 were applied to label the cis-diol metabolites. Based on the chemical isotope labeling, cis-diol metabolites were easily recognized from urine samples. Influence of BPA exposure on these metabolites was investigated by comparing different doses of BPA administration on rats. Analytes showed noticeable difference were highlighted. Pathway analysis indicated that galactose metabolism, nucleoside and its analogues metabolism were disturbed. The derivatization method was extended to quantify nucleotides in plasma samples. According to the specific physical-chemical properties of nucleotides, the method was improved to fit the requirement of analysis by using 1,1-Dimethoxycyclohexane (DMCH) as derivatization agent and formic acid (FA) as catalyst. Tip micro-columns packed with TiO2 were used for selective adsorption of nucleotides in the plasma. Then in-situ derivatization were carried out to change the polarity of targeted compounds. LC-MS analysis of the derivatization products were employed without using ion-pairing reagents. This method exhibited a high selectivity for the extraction of nucleotides. After derivatization, retention of nucleotides on reversed-phase C18 column was improved. Complete separation of nucleotides with the same base was achieved. The peak shape was symmetrical and the tailing was eliminated by using high pH mobile phase. The method settled the problems of nucleotide detection, which were poor retention, trailing, in-source fragmentation and contamination of ion-pairing reagents. The quantitative method was successfully applied to determine the content of nucleotides in plasma samples of rats exposed to BPA. It was simple and fast, as well as good selectivity and stability. It could be extended to detection of other phosphorylated metabolites with similar structure. To our best knowledge, it was the first time to employ derivatization methods to detect cis-diol-containing metabolites. The methods decreased the matrix effects of complex biological samples, and also decreased the polarity of cis-diol-containing metabolites. The changes of properties not only improved the chromatographic separation, but also enhanced the MS intensities. The methods overcame the problems of cis-diol-containing metabolite detection on reversed-phase column. They were successfully applied to study the changes of cis-diol-containing metabolites of HCC and toxic effects of BPA exposure. The method might be extended to determine other cis-diol-containing metabolites in urine samples as well as in cells, tissues and plasma samples. It might be valuable for the understanding of the roles of cis-diol-containing metabolites in in cell metabolism.

O trabalho realizado teve como objetivo principal o projeto, a construção e os testes de um sistema de vácuo dedicado a técnica de Espectrometria de Massa do íon Secundário (SIMS), a ser utilizado na caracterização estrutural de heteroestruturas semicondutoras. O sistema (câmaras e conexões) foi totalmente desenvolvido nas dependências do IFSC-USP, sendo acoplado a este um conjunto de elementos comerciais, tais como: visores de Ultra-Alto Vácuo (UHV), medidores, canhão de íons, válvulas, bombas (mecânica, turbo e iônica) e espectrômetro de massa. Testes realizados mostraram que o sistema de vácuo principal (o sistema todo é composto por duas câmaras de vácuo: uma principal e uma de introdução) pode atingir pressões da ordem de 10-10 Torr, necessária para a implementação de técnicas de análise de superfície. Além disto, o sistema mostrou-se versátil para a troca de amostras (uso continuo) e de fácil operação. Além de instrumentação para UHV, os resultados obtidos são promissores no sentido de proporcionar uma economia nos custos para futuras aquisições de sistemas para analise de superfícies / The principal purpose of this work was the project, construction and test of a vacuum system dedicated to Secondary Ion Mass Spectroscopy (SIMS), for the structural characterization of semiconductor heterostructures. All the system components were developed at the IFSC-USP, to which were connected commercial vacuum elements, such: Ultra-high Vacuum (UHV) visors, sensors, ion gun, vacuum pumps (mechanical, turbo and ionic) and mass spectrometer. The results showed that the main chamber (the system is composed by two chambers: main chamber and the load-look) is able to reach pressures of 10-10 Torr, important for surface analyses. Also the system showed versatility to change the samples and easy operation. The results for UHV instrumentation developed are promising, and these results can be used to decrease the cost of surface analysis systems which can be implemented in the future