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abstract: Cancer claims hundreds of thousands of lives every year in US alone. Finding ways for early detection of cancer onset is crucial for better management and treatment of cancer. Thus, biomarkers especially protein biomarkers, being the functional units which reflect dynamic physiological changes, need to be discovered. Though important, there are only a few approved protein cancer biomarkers till date. To accelerate this process, fast, comprehensive and affordable assays are required which can be applied to large population studies. For this, these assays should be able to comprehensively characterize and explore the molecular diversity of nominally "single" proteins across populations. This information is usually unavailable with commonly used immunoassays such as ELISA (enzyme linked immunosorbent assay) which either ignore protein microheterogeneity, or are confounded by it. To this end, mass spectrometric immuno assays (MSIA) for three different human plasma proteins have been developed. These proteins viz. IGF-1, hemopexin and tetranectin have been found in reported literature to show correlations with many diseases along with several carcinomas. Developed assays were used to extract entire proteins from plasma samples and subsequently analyzed on mass spectrometric platforms. Matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometric techniques where used due to their availability and suitability for the analysis. This resulted in visibility of different structural forms of these proteins showing their structural micro-heterogeneity which is invisible to commonly used immunoassays. These assays are fast, comprehensive and can be applied in large sample studies to analyze proteins for biomarker discovery. / Dissertation/Thesis / M.S. Biochemistry 2012

Mass spectrometry is a versatile, powerful analytical tool for chemical and biomolecule identification, quantitation, and structural analysis. Tandem mass spectrometry is key component in expanding the capabilities of mass spectrometry beyond just a molecular weight detector. Another key component is the discovery of electrospray ionization allowing not only liquid samples to be ionized but also generation of multiply charged ions enabling mass spectrometry analysis of large biomolecules. The fragmentation pathway of ion during tandem mass spectrometry is highly dependent on the nature of the ion as well as the form of dissociation technique employed. To-date, no single form of ion or dissociation method can provide all the structure information needed; therefore, it is common to use multiple forms of ions, different charge carrier or modifications, with a variety of other dissociation techniques to generate complimentary information. Practically, it is not always easy to generate the desired form of ion via ionization methods and is one of the limitations. Gas-phase ion/ion reactions provide an easy approach in manipulation of ions, either through changing the ion type or covalent modifications, in the gas-phase with the goal of enhancing the capabilities of mass spectrometers for either molecular weight or structural analysis. In this dissertation, studies of new gas-phase ion/ion chemistry for biomolecules such as carbohydrates and phopho/sulfopeptides were performed, and exploration into mass spectrometry analysis of IgGs is discussed. <br><div> Ion/ion reactions with carbohydrates were investigated with the goal of finding a charge-transfer or covalent modification reaction which can increase the structural information of carbohydrates upon tandem mass spectrometry. No luck was achieved with charge transfer ion/ion reactions which increased the overall fragmentation information in tandem mass spectrometry. Novel gas-phase covalent chemistry was discovered where alkoxides were found to form ester and ethers. It was also discovered the aldehyde functional group at the reducing end of carbohydrates are susceptible to Schiff-base modifications. Schiff-base has been previously reported in peptides and this is the first time it has been discovered for carbohydrates.</div><div> In the next project a gas-phase approach for the rapid screening of polypeptide anions for phosphorylation or sulfonation based on binding strengths to guanidinium-containing reagent ions was developed. The approach relies on the generation of a complex via reaction of mixtures of deprotonated polypeptide anions with dicationic guanidinium-containing reagent ions and subsequent dipolar DC collisional activation of the complexes. The relative strengths of the electrostatic interactions of guanidinium with deprotonated acidic sites follows the order carboxylate</div><div> Hyaluronic acid, a linear carbohydrate polymer with repeating units of D-glucuronic acid and N-acetyl D-glucosamine, was found to exhibit unique properties in its electrospray ionization mass spectrum that was never seen before. Electrospray of hyaluronic acid in aqueous solution in the negative polarity presented an incredibly intriguing mass spectrum, which we termed “emerald city” consisting of max charge or max charge-1 anions of hyaluronic acid. This is the first biomolecule observed to have the capability to deprotonate at every acid site that is possible. These set of highly charge anions exhibits unique characteristics upon use as a charge inversion reagent to charged invert multiply protonated proteins. A max of thirty-three protons was transferred when myoglobin 24+ was charge-inverted to a max charge state of 9- in the negative mode. Further research should be conducted to fully understand this phenomenal and its possible utilities.<br></div><div> Lastly, mass spectrometry analysis of monoclonal antibodies was performed. Monoclonal antibodies are 150 kDa sized protein complexes and is a major area of interest for pharmaceutical industry. Mass spectrometry analysis of big proteins is an emerging area for mass spectrometry and is quite different compared to small and medium molecule analysis on the mass spectrometer. Detailed in the last chapter are methods developed for sample cleanup of immunoglobulin G as well as the application of q2 DDC for removal of loosely bound adducts to achieve sharper peaks in the mass spectrum. Studies of protein denaturation was also conducted with methods such as circular dichroism and differential ion mobility also employed. And finally, a photochemical reaction setup was shown to cleave twelve out of sixteen total disulfide bonds in the immunoglobulin G within seconds compared to traditional solution phase reactions which can take hours.<br></div>

<p> </p> <p>  Mass spectrometry (MS) plays an important role in nanomaterials research by facilitating the discovery of superatomic clusters and fullerenes, enabling the identification of atomically substituted clusters, and contributing to understanding mechanisms of cluster formation. In this dissertation, we used different mass spectrometry methods as well as ion soft-landing to address some of the ongoing challenges in cluster science. The first challenge is to extend the atom-by-atom substitution method, which is a promising strategy for designing new cluster-based materials, to a wider range of molecular clusters. Due to challenges associated with the synthesis, purification, and crystallization, this approach has been achieved only for a handful of gold and silver clusters. We extended this approach to enable the substitution of the first-row transitional metals into the core of Co6S8(PEt3)6 cluster, a well-defined metal chalcogenide superatomic cluster and a popular building block for designing novel 2D materials. This cluster is widely used in energy and electronic applications and is an excellent model system for computational studies of cluster-based materials. High-resolution MS analysis identified the formation of Co5MS8(PEt3)6+ (M=Mn, Fe, Ni) clusters, indicating that only Mn, Fe, and Ni atoms can be incorporated into the Co6S8 core using our synthetic method. A combination of mass spectrometric analysis and theoretical studies reaved that each heteroatom has different impact on the relative stability, core-ligand interaction, as well as optical, magnetic, and electrochemical properties of the cluster. </p> <p>Another challenge in the cluster science addressed in this work is the controlled activation of fully ligated clusters by ligand removal. Conventional activation methods such as thermolysis or chemical treatment do not provide sufficient control of the number of the removed ligands and often suffer from sintering of NCs as a result of excessive ligand removal and degradation of the destabilized NC cores. Using a specially-designed ion soft-landing instrument, we achieved a controlled removal of one or two phosphine ligands form the synthesized cobalt sulfide clusters using collision-induced dissociation (CID). The resulting fragments were mass selected and soft-landed onto surfaces. We found out that the reactivity of the fragment ions on surfaces may be controlled by altering the composition of the cluster core and ligand binding energy to the cluster. Although some of the fragments formed by removing one ligand including Co5FeS8(PEt3)5+ and Co6S8L(PPh3)5+ remain unreactive on surfaces, other fragments including Co6S8(PEt3)5+, Co5NiS8(PEt3)5+, and Co6S8(PEt3-xPhx) (x=1-2) undergo selective dimerization.  We observe that the reactivity of fragment ions produced by removing one surface ligand is controlled by the relative stability of the corresponding precursor ions towards fragmentation. In particular, fragment ions generated from more stable precursors undergo a selective dimerization reaction. In contrast, fragment ions produced from the least stable precursors remain largely unreactive on surfaces. In addition, we found that fragments generated by removing two surface ligands are highly reactive and undergo several nonselective reactions. This study demonstrates that fragment ions are unique building blocks that may undergo selective reactivity on surfaces to generate compounds that are difficult to prepare using conventional synthetic methods. We believe that the controlled preparation of fragment ions using ion soft-landing is a generalizable method to activate wide range of ligated nanoclusters which opens a direction for materials design and innovation.</p> <p>Finally, soft-landing of well-characterized redox-active polyatomic anions, PW12O403- (WPOM), was carried out to explore the distribution of pure mass selected anions on semiconducting vertically-aligned TiO2 nanotubes, which were used as a model system for 3D semiconductive materials. Energy dispersive X-ray (EDX) mapping analysis revealed that WPOMs form micron-size aggregates on top of the TiO2 NT and only penetrates 1-1.3 µm into the 10 µm-long nanotubes. This aggregation is attributed to the high resistance of TiO2. This is different from what we see on conductive substrates such as carbon nanotubes (CNTs) where a uniform distribution of ions is typically observed. This study provides valuable insight into the functionalization of porous semiconducting surfaces using mass selected ions for applications in energy storage and electronics.</p>

<p> An attempt was made to measure the yields of the stable cadmium isotopes produced in the thermal-neutron fission of 233U and 235U using a solid-source mass spectrometer. The results for 235U fission indicate that there is structure in the mass-yield curve for the region studied which takes the form of a depression around masses 112-114. The origin of this structure is discussed in terms of the various mechanisms which have been proposed to explain the nature of the mass distribution in the symmetric region.</p> <p> The 233U study was unsuccessful because of the experimental difficulties encountered, primarily the interference from terrestrial cadmium and the low recovery of the fission products. </p> / Thesis / Master of Science (MSc)

Neste estudo foram avaliados procedimentos analíticos comparativos para o preparo de três tipos de amostras agroindustriais (rochas, cafés e antibióticos) na determinação de elementos inorgânicos pelas técnicas de espectrometria de absorção atômica em chama (FAAS), espectrometria de emissão atômica em chama (FAES) e espectrometria de absorção atômica em forno de grafite (GFAAS). Seis procedimentos de decomposição de rochas foram estudados e classificados em 3 grupos: GRUPO I: bombas de teflon; GRUPO II: bloco digestor; GRUPO III: forno de microondas convencional. Os elementos Cu, Fe, Mn e Zn foram quantificados pela técnica FAAS em duas amostras comerciais e dois materiais de referência padrão de rochas. Os melhores resultados foram obtidos para os procedimentos que empregaram forno de microondas convencional usando-se HF na mistura ácida. Os teores de 9 nutrientes foram determinados em 35 amostras de café (verde e torrado) por FAAS (Ca, Mg, Fe, Cu, Mn e Zn), FAES (Na e K) e pelo método Kjeldahl (N). As amostras foram preparadas por dois procedimentos de decomposição via úmida, um deles empregando bloco digestor e V2O5 como catalisador e o outro forno de microondas convencional. A exatidão dos métodos foi verificada pela análise de três materiais de referência padrão de folhas. Um teste foi realizado moendo-se uma das amostras de café verde em dois tipos de moinho (criogênico e tipo Willye) para avaliar possíveis contaminações. Contaminação com Fe foi observada com o uso do moinho tipo Willye. Outro teste constou da determinação desses 9 nutrientes em uma amostra de café verde com casca. Altos teores de Na, Ca, K e Fe foram encontrados na casca do café verde, confirmando assim a possibilidade de reaproveitamento desses resíduos em processos de adubação e na produção de ração animal. Análises quimiométricas aplicando-se PCA e HCA mostraram que os elementos Na, K, Ca, Mg e Fe foram os principais responsáveis pela discriminação entre as amostras de café verde e torrado. Um método foi desenvolvido para a determinação simultânea de Cd e Pb por GFAAS em antibióticos sólidos usados na fermentação da cana de açúcar. Seis amostras de antibióticos sólidos comerciais foram analisadas injetando-se 20 µL das amostras decompostas em meio ácido (forno de microondas convencional) dentro da plataforma de grafite pré-tratada com W seguido de 5 µL do modificador químico Pd 1000 mg L-1. As amostras foram solubilizadas por dois procedimentos: 1) 3 mL HNO3 conc. + 2 mL H2O2 30% (v/v) e, 2) 4 mL HNO3 20% (v/v) + 1 mL H2O2 30% (v/v). As temperaturas de pirólise e atomização do programa de aquecimento do atomizador foram fixadas em 600oC e 2200oC, respectivamente. As massas características calculadas para a determinação simultânea foram 1,6 pg Cd e 41,7 pg Pb. Limites de detecção (LOD) baseados na absorbância integrada foram 0,02 µg L-1 Cd e 0,7 µg L-1 Pb e os desvios padrões relativos (n = 10) para Cd e Pb foram 5,7% e 8,0%, respectivamente. As recuperações de Cd e Pb adicionadas às amostras digeridas variaram de 91% a 125% (Cd) e 80% a 112% (Pb). / Analytical comparative procedures for the preparation of three types of agro industrials samples (rocks, coffees and antibiotics) were evaluated in the determination of inorganic elements by flame atomic absorption spectrometry (FAAS), flame atomic emission spectrometry (FAES) and graphite furnace atomic absorption spectrometry (GFAAS) techniques. Six decomposition procedures of rocks were studied and classified in 3 groups: GROUP I: wet digestion in teflon bombs; GROUP II: wet digestion in digester heating block; GROUP III: wet digestion in conventional microwave oven system. The elements Cu, Fe, Mn and Zn were quantified by FAAS in two commercial samples and two standard reference materials of rocks. The best results were obtained for the procedures that employed conventional microwave oven system using HF in the acid mixture. The contents of 9 nutrients were determined in 35 coffee samples (green and roasted) by FAAS (Ca, Mg, Fe, Cu, Mn and Zn), FAES (Na and K) and Kjeldahl method (N). Samples were prepared by two different wet digestion decomposition procedures; one of them employing digester heating block and the catalyst V2O5 and the other one employing a conventional microwave oven system. The accuracy of the methods was checked after analysis of three leave standard reference materials. A test was done in a green coffee sample ground in two types of mill (cryogenic and Willye) to evaluate possible contaminations. Contamination by Fe was observed with the use of Willye type mill. Another test was the determination of these 9 nutrients in an unpulped ripe cherry. High levels of Na, Ca, K and Fe were encountered in the green coffee rinds, which can be used as a complement in manuring process and for animal ration production. Chemometric analysis using PCA and HCA showed that Na, K, Ca, Mg, Cu and Fe were the principal elements to discriminate samples between green and roasted coffee. A method has been developed for the simultaneous determination of Cd and Pb in antibiotics used in sugar-cane fermentation by GFAAS. Six samples of commercial solid antibiotics were analyzed by injecting 20 µL of digested samples (closed microwave oven system) into the pretreated graphite platform with W followed by 5 µL of 1000 mg L-1 Pd chemical modifier. Samples were solubilized by two procedures: 1) 3 mL HNO3 conc. + 2 mL H2O2 30% (v/v) and, 2) 4 mL HNO3 20% (v/v) + 1 mL H2O2 30% (v/v). The pyrolysis and atomization temperatures of the heating program of the atomizer were fixed at 600oC and 2200oC, respectively. The calculated characteristic masses for the simultaneous determination were 1.6 pg Cd and 41.7 pg Pb. Limits of detection (LOD) based on integrated absorbance were 0.02 µg L-1 Cd and 0.7 µg L-1 Pb and the relative standard deviations (n = 10) for Cd and Pb were 5.7% and 8.0%, respectively. The recoveries of Cd and Pb added to the digested samples varied from 91% to 125% (Cd) and 80% to 112% (Pb).

This work has two interlocking themes. It is primarily concerned with the development of precise, broad band, Fourier transform spectrometric techniques for the determination of the optical constants of solids at millimetre and submillimetre wavelengths. One such technique cannot easily accomodate the wide range of optical constants found in solids, leading to specimens which range from the virtually transparent to the virtually opaque, and it was therefore necessary to develop transmission and reflection techniques. The intercomparison of these techniques, their particular experimental difficulties and susceptibilities to random and systematic error, was performed by using each method to determin'the optical constants of soda lime silica glass over as wide a spectral range as possible. Previous to this work there had been no systematic study of this important material at these wavelengths and,thus, this investigation gave the second theme of this work, the quantitative determination of the optical constants of soda lime silica glass at millimetre and submillimetre wavelengths. The results of this study are presented in four chapters. First, in chapter 5, power transmission Fourier transform spectrometry has been used to investigate the spectral variation of the optical constants of the glass between 3 and 50cm, using an analysis of channel spectra to give the refractive index. This chapter also contains the results of measurements made with a grating instrument between 1000 and k000cm. Secondly, in chapter 6, the results of the first direct determinations of the optical constants by dispersive transmission Fourier transform spectrometry are presented and shown to indicate the presence of a hitherto unknown loss process below 20er that is tentatively associated with a similar process previously observed in fused silica. Thirdly, in chapter 7, dispersive reflection Fourier transform spectrometry has been used to determin the optical constants up to 360em, well into the midinfrared region of opacity of this glass. The results of chapters 6 and 7 provide the first quantitative description of the optical constants of this glass betweem 3 and 360em Finally, in chapter 8, the dispersive reflection measurements are extended to simple binary and ternary silicate glasses, and these results used to identify the contributions to the mid-infrared absorption of the metal ions associated with the various metal oxide additives of the glass. From this it was possible to account for all of the absorption in soda lime silica glass as a superposition of contributions from the near-infrared bands of the SiO1 network of the glass and the four main metal ions.

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