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<p>By studying the boron isotope composition of marine carbonates, the effectiveness of the tracer to reconstruct the ancient seawater pH and, in turn, atmospheric pCO₂ can be assessed. The boron isotope-pH proxy relies on the hypothesis that only B(OH)₄^-, which has a known boron isotope composition with respect to pH, is incorporated into the carbonate crystal lattice. This research synthesized inorganic aragonite from a range of stable pH values to quantify the dependence of aragonite <em>δ</em>¹¹B on the pH of the precipitating solution. The increasing boron isotope composition of aragonite with increasing pH is consistent with sole incorporation of B(OH)₄^- into the carbonate. The sensitivity of the acid dissociation and isotope equilibrium constants make it difficult to confirm whether or not B(OH)₄^- is the only species contributing to the boron isotope composition of aragonite. The relationship between the boron isotope composition of marine carbonates and ocean pH has wide appeal and, if properly understood, could provide tremendous insight into the history of Earth’s climate.</p> <p>This research also evaluated carbon isotope fractionation between aragonite and dissolved inorganic carbon (DIC) in high ionic strength systems at 25 °C and found it to be consistent with carbon isotope fractionation in low ionic strength environments. The analysis of various isotopic compositions within this study led to the development of new methodology to simultaneously measure the oxygen isotopes of water and carbon isotopes of DIC from small solution samples using continuous flow isotope ratio mass spectrometry (CF-IRMS).</p> / Master of Science (MSc)

Alkylphenol ethoxylates (APEOs), alcohol ethoxylates (AEOs), and alcohol propoxylates (APOs) are non-ionic surfactants used in daily care products and detergents. They are formed as an oligomeric series with a varying distribution, which determines their commercial application. The goal of the research performed was the development of sample characterization methods for non-ionic surfactants utilizing supercritical fluid chromatography (SFC) under mild instrument operating conditions. The aryl group present in APEOs allowed ultraviolet (UV) detection, with an equal molar response for oligomers, allowing average molar oligomer values to be calculated. APEOs were separated by ethoxylate unit via SFC-UV as well as normal phase HPLC-UV employing packed columns. Stationary phase and column length were varied in the SFC setup to produce the most favorable separation conditions. Fractions from SFC runs of APEOs were collected and analyzed by flow injection analysis electrospray ionization mass spectrometry (FIA-ESI-MS) to identify fraction composition. SFC provided shorter retention times with similar resolution as HPLC for separation of APEOs and consumed a smaller amount of organic solvent. AEOs and APOs lack functionality capable of absorbing UV light outside the UV cut-off of normal organic solvents. SFC was able to separate AEOs and APOs derivatized as trimethylsilyl ethers (TMS) with pure CO2 with detection at 195 nm. The instrumental conditions, however, needed for separation necessitated high temperature and high CO2 pressure. Derivatization of alcohol polyether samples with an UV absorbing agent was achieved with phenylated disilazane-chlorosilane mixtures forming phenylsilylethers detected at 215 nm. Use of an organic solvent-modified CO2 mobile phase afforded lower pressure and temperature conditions for oligomer separation. The use of polar embedded alkyl phases combined with use of organic modified CO2 produced good resolution between oligomers. Better peak shape and shorter retention times were realized with methanol-modified CO2 than acetonitrile-modified CO2. Peak assignments were made via SFC coupled with ESI-MS detection in the positive ion mode. SFC-UV and SFC-ESI-MS data were jointly used for calculation of average molar oligomer values. Proton nuclear magnetic resonance (1H-NMR) analysis of non-derivatized samples was performed to determine average molar oligomer values and was used for comparison with values calculated from SFC-UV data. / Ph. D.

The positive and negative ion mess spectra and appearance potentials were determined for the following compounds: benzil, 4-methylbenzil, 4- methoxybenzil, 4-methylmercaptobenzil, 4-dimethylaminobenzil, 4-chlorobenzil, 4—nitrobenzil, 4,4'-dimethylbenzil, 4,4'-dimethoxybenzil, 4,4'- bis(methylmercapto) benzil, 4,4'-bis(dimethylamino)benzil, 4,4'-dichlorobenzil, and 4,4‘-dinitrobenzil. Metastable data were used to establish that the fragmentation mechanism for positive ions involves cleavage between the carbonyls followed by loss of CQ. Calculated appearance potentials are compared wilh those generated experimentally. The appearance potentials of the substituted benzoyl ions exhibit substituent effects correlatable with the Hammett σ, but no substituent effect is demonstrated for the unsubstituted benzoyl. Loss of CO by the benzoyl ion does not appear to involve substituent effects but required 2.5-4.0 eV excess energy above that required to form the precursor. The nitro-substituted benzils fragment by two competing mechanisms, that described supra and the loss of NO⋅ following rearrangement of the substituent. The base peak in all negative ion spectra at 50 eV was the parent-molecule jon. Benzil, the methyl-substituted benzils, and the dimethyl-amino- substituted benzils form no daughter ions. The methoxy-substituted benzils and the nethyimercapto-substituted benzils formed a daughter jon by loss of CH₃⋅ from the parent, The echloro-substituted benzils fragment to produce Cl⁻. The nitro-substituted benzils lose NO⋅, apparently following a rearrangement Similar to that observed for positive ions, and also fragment to lose NO⁻ in low abundance. Appearance potential data indicate that all parent-molecule ions are formed by electron capture, and all fragments by dissociative-attachment. The electron-withdrawing substituents cause a broadening of the ionization efficiency curve for the parent-molecule ion; all other substituents have no effect on the curves' shape. Daughter ion ionization efficiency curves are broader than the parent-molecule i.e. curves, and the i.e. curves for the daughter ions from the methoxy-substituted benzils appear to be conmposits of at least two curves. NO⁻ and Cl⁻ are of low abundance, with onset at 2.2 eV and 5.5 eV respectively above SF¯₆. The most abundant negative daughter ions are resonance stabilized; Cl⁻ and NO⁻ are formed by electron pairing at an electronegative site. Negative ions are not formed with sufficient excess energy to fragment by the same mechanism as positive ions, with the exception of the anomalous nitro-substituted mechanism. Polarographic half-wave potentials were compared to negative ion mass spectrometric data. Benzils with electron-donating substituents fit a plot of E_1/2 vs.o. Substituent effects for these benzils are additive, as indicated by comparison of Ag for one and two substituents to that for benzil. Chloro-substituted and nitro-substituted benziils have second waves at only 0.3 to 0.4 v more negative than the first wave, in contrast to 0.6 to 0.8 v differences for the other benzils. Loss of a substituent, as observed in the negative ion mass spectra, is suggested as an explanation E_3/4 - E_1/4 values were calculated and indicate that waves in the region 1.0 to 1.4 v are probably reversible. / Ph. D.

<p dir="ltr">Mass spectrometry (MS) is a powerful tool for analyzing complex mixtures, offering valuable information regarding identification and quantification for laboratory research, both fundamental and applied. Manipulating ions using electrostatic and electrodynamic fields in a vacuum environment, MS is inherently fast and capable of generating diverse structural information. However, the potential speed of MS analysis is often hindered due to the necessity of separating complex mixtures prior to injection into the mass spectrometer. In this dissertation, we demonstrate methods to improve the speed of mass spectrometric mixture analysis at every stage, from ionization to ion trapping, and eventually to data analysis.</p><p dir="ltr">One solution to the challenge of increasing speed is ambient ionization using nESI coupled with microdroplet-based reaction acceleration. During the travel of the microdroplet from the ionization source to the inlet of the mass spectrometer, a derivatization reaction was shown to increases the signal of compounds with a targeted functional group from the mixture within milliseconds. This eliminated the need for time-consuming chromatographical separation, allowing for class-based mixture analysis (<i>e.g.</i> phenolic compounds, chapter 2). This accelerated chemical reaction mechanism was studied using the Katritzky reaction and a large surface-to-volume ratio (chapter 3). It is suggested that acceleration is associated with an electric double layer, which creates a strong electric field near the air-liquid interface.</p><p dir="ltr">The second part of the discussion on speeding up mixture component identification uses a modified ion trap and a new scan type: two-dimensional tandem mass spectrometry (2D MS/MS). In this experiment, MS/MS data of all precursors is recorded without isolation, thereby allowing characterization of each molecule in the mixture using a single ion injection. The generated data is stored in an image format where one dimension represents the precursor domain while the other dimension represents the product <i>m/z</i> values. This allows for the structural interpretation of all mixture components from a single spectrum. Applications of this technology include developing point-of-care assays to analyze patient blood HIV drug levels in less than five minutes (chapter 5 and 6) and the <i>in-situ</i> analysis of sulfonamides in milk in less than one minute (chapter 7) using portable mass spectrometers.</p><p dir="ltr">The third improvement is focused on novel data analysis techniques. In one case, the effects of an accelerated reaction on a complex mixture were visualized using subtracted 2D MS/MS spectra, which provided an additional layer of information, speeding up the processing and interpretation of an unknown mixture (chapter 8). In another case, raw 2D MS/MS spectra were formatted and processed for machine learning analysis, which was used to classify and identify eight different bacteria based on lipid region profiles within seconds.</p>

Cancer is characterized by a deregulation of the cell cycle resulting in abnormal proliferation of cells that can bypass tightly regulated molecular checkpoints. Breast cancer is the most common cancer diagnosed in women, ~70% of cases displaying an estrogen receptor positive (ER+) phenotype. The aim of the present work was to generate a comprehensive overview of the biological mechanisms, molecular pathways and specific proteins involved in cell cycle progression in ER+ breast cancer cells. We focused on the G1-to-S phase transition of the cell cycle because major differences in cell proliferation mechanisms between normal and cancerous cells are observed at this point. We developed a large-scale proteomics strategy to enable the comparison of MCF-7 ER+ (cancer) and MCF-10A (non-tumorigenic) epithelial breast cells. Samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) followed by a label-free quantitation approach, i.e., spectral counting, for differential protein expression analysis. The study was divided into three distinct parts: 1) qualitative profiling of MCF-7 cells arrested in the G1-phase and released into the S-phase of the cell cycle, 2) differential expression profiling of MCF-7 cells in G1 and S, and 3) differential expression profiling of the G1-phases of MCF-7 and MCF-10A cells. The qualitative evaluation of MCF-7 proteomic data resulted in the identification of >2700 proteins (p-score<0.001). A large number of these proteins were involved in cell cycle relevant processes, being representative of all hallmarks of cancer. Differential expression analysis of the MCF-7 G1 and S-phases resulted in the identification of >250 proteins with roles in DNA repair, transcription, translation, chromatin maintenance and signaling. The MCF-7/MCF-10 comparison revealed that major cellular processes that require DNA access, such as the ones identified in the MCF-7 analysis, are up-regulated in the nucleus of MCF-7 cells during starvation, possibly allowing these cancerous cells to bypass the restriction point. Several proliferative and anti-proliferative markers were identified in both MCF-7 and MCF-10A cells. / Ph. D.

The discovery of cancer biomarkers is crucial in the clinical setting to facilitate early diagnosis and treatment, thereby increasing survival rates. Proteomic technologies with mass spectrometry detection (MS) have the potential to affect the entire spectrum of cancer research by identifying these biomarkers. Simultaneously, microfabricated devices have evolved into ideal analysis platforms for minute amounts of sample, with promising applications for proteomic investigations and future biomarker screening. This thesis reports on the analysis of the proteomic constituents of the MCF7 breast cancer cell line using a shotgun 2-D strong cationic exchange/reversed phase liquid chromatography electrospray ionization tandem mass spectrometry (SCX/RP-LC-ESI-MS/MS) protocol. A series of optimization strategies were performed to improve the LC-MS experimental set-up, sample preparation, data acquisition and database searching parameters, and to enable the detection and confident identification of a large number of proteins. Over ~4,500 proteins were identified using conventional filtering parameters, and >2000 proteins using a combination of filters and p-value sorting. Of these, ~1,950 proteins had p<0.001 (~90%) and more than half were identified by &#8805; 2 unique peptides. About 220 proteins were functionally involved in cancer related cellular processes, and over 100 proteins were previously described in the literature as potential cancer markers. Biomarkers such as PCNA, cathepsin D, E-cadherin, 14-3-3-sigma, antigen Ki-67, TP53RK, and calreticulin were identified. These data were generated by subjecting to mass spectrometric analysis ~42 µg of protein digest, analyzing 16 SCX peptide fractions, and interpreting ~55,000 MS2 spectra. Total MS time required for analysis was 40 h. Selective SCX fractions were also analyzed by using a microfluidic LC platform. The performance of the microchip LC was comparable to that obtained with bench-top instrumentation when similar experimental conditions were used. The identification of 5 cancer biomarkers was enabled by using the microchip LC platform. Furthermore, this device was also capable to analyze phosphopeptides. / Master of Science

Proteomics is defined as a tool to explore how proteins control and regulate important molecular and physiological processes. Further, peptide-centric approaches, or bottom-up methods, provide more comprehensive coverage of a proteome compared to whole-protein approaches. This body of work assesses the technical feasibility of several bottom-up proteomics technologies applied to Arabidopsis thaliana, including gel-based methods, those that require peptide derivitization, and those that do not. Selected-reaction monitoring (SRM) for targeted proteomics, and data-independent acquisition (MSE) was also evaluated. In addition to assessing the capabilities of these technologies, we then applied them to the context of uncovering new insights into the flavonoid biosynthetic pathway and the auxin and ethylene signaling pathways. Chapter one provides background information related to secondary metabolism, phytohormone signaling, and the status of proteomics in plants. In Chapter 2 and Appendix A, we establish the methodology to apply traditional and DiGE-based 2D-GE strategies to global proteomics in Arabidopsis. Our results suggest that while 2D-GE is applicable to Arabidopsis, there are practical and conceptual limitations that must be understood. Further, our results suggest that pertubations in the flavonoid pathway do not affect the abundance of proteins in Arabidopsis seedlings, roots, or flowers that can be studied using 2D-GE and DiGE. Additionally, we demonstrated the first parallel comparison of the effects of auxin and ethylene on the Arabidopsis root proteome and observed no overlap among the proteins regulated by the two phytohormones, at least for the most abundant proteins observed by 2D-GE. Chapter 3 explores the efficacy of selected reaction monitoring for relative peptide quantification in Arabidopsis roots. Our results suggest that while the technology parallels application in yeast and humans, there are substantial analytical challenges that much be addressed. In Chapter 4 we explore the MSE data acquisition scheme for global proteomics in Arabidopsis. We observe that treatment with exogenous auxin affects the abundance of many proteins representing diverse biological processes. Interestingly, we observe minimal overlap among genes and proteins regulated by exogenous auxin. Appendix B explores the efficacy of iTRAQ labeling for relative peptide quantification in Arabidopsis roots. / Ph. D.