Oligosaccharidoses are Lysosomal Storage Disorders (LSDs) that result from mutations in genes encoding exoglycosidases, leading to accumulation of unmetabolized N-linked oligosaccharides within lysosomes. Age at onset and rate of disease progression vary among patients. Diagnosis based solely on clinical presentation is often challenging because of overlapping clinical symptoms between these disorders. The aim of this research is to use tandem mass spectrometry (MS/MS) to establish a multiplex method to measure exoglycosidase activities, in dried blood spots (DBS), involved in the degradation of N-linked oligosaccharides using natural substrates. Current fluorometric assays for each exoglycosidase using specific 4-methylumbelliferyl (4MU) substrates allow enzyme activities to be determined separately from a variety of human tissue sample types. A universal buffer was established by comparing these assay conditions to allow multiplexing of the exoglycosidases in a single vial. Initial attempts to develop an enzyme activity assay using disaccharides as the starting substrate and by monitoring unique monosaccharide products by MS/MS after exposure to an enzyme source from cultured skin fibroblasts were unsuccessful due to interfering endogenous hexose isomers. Taking another approach, multiplexing was successfully demonstrated for beta-Galactosidase and beta-Hexosaminidase using alternative substrates. 4MU and paranitrophenol (PNP) conjugated to particular monosaccharides allowed 4MU and PNP products to be measured and enzyme activities to be calculated. Here, we provide a proof of principle that MS/MS technology can allow simultaneous multiplexing of several enzyme activities using distinctive starting substrates. A multiplex assay for the remaining exoglycosidases can still permit the development of an Oligosaccharidoses screening test to assist clinical diagnosis.

Capillary electrophoresis (CE) is the workhorse of countless analytical laboratories and is used routinely in various industries including pharmaceutical, forensic and clinical applications. Basically, CE is a method for separating charged molecular species in a buffer-filled capillary by the application of an electric field; the analytes move from one end of the capillary to the detector at the other end at speeds determined by their charge, size and shape. Generally, in free solution CE uniformly charged polyelectrolytes (such as DNA) are free-draining, meaning that their speed is independent of their size. Hence, until recently, a gel or other sieving medium has been necessary for the separation of polyelectrolytes; however, modifying uniformly charged polymers on the molecular level, via conjugation to uncharged polymers, allows for separation in free solution CE. In this thesis, advancements to the theory of free solution electrophoresis of polyelectrolytes, in particular, to the theories for two new free solution electrophoresis methods relying on conjugation, are presented. The first method, called End Labelled Free Solution Electrophoresis (ELFSE), can be used to sequence DNA, a negatively charged polymer in solution. Two different means of improving the resolution of ELFSE are predicted, one based on the molecular end effect, the other based on using a controlled electro-osmotic flow. In addition, a theory for the segregation of the DNA and label coils in ELFSE is presented. The second method is called Free Solution Conjugate Electrophoresis (FSCE); it allows for characterization of a sample of neutral polymers differing in length. The relevant theory, developed herein, elucidates how to accurately determine the molar mass distribution of the sample through FSCE measurements. In addition, supporting theories are developed that clarify the correct equation for the diffusion coefficient of molecules undergoing free solution electrophoresis, as well as illustrate that under ideal conditions, a viscosity gradient within the capillary serves only to decrease resolution and hence can not be used to improve performance. These theoretical studies constitute the six articles presented in this thesis. In addition, a comprehensive review article covering the development of ELFSE over the last decade, the theoretical concepts used to predict the ultimate performance of ELFSE for DNA sequencing, and the technological advances that are needed to speed the development of competitive ELFSE-based sequencing and separation technologies, is given in Appendix A. The predicted improvement in ELFSE resolution based on the end effect theory was also proven experimentally; the article with these findings is provided in Appendix B.

Electrospray ionization (ESI) and matrix assisted laser desorption-ionization (MALDI) mass spectrometry were used to determine the composition (monomer ratios) and structure (end group analysis), relative to 1H NMR spectroscopy and theoretical predictions, for three different copolymers: poly(butyl acrylate/vinyl acetate) (PBA/PVAc), poly(methyl methacrylate/vinyl acetate) (PMMA/PVAc) and poly(butyl acrylate/methyl methacrylate) (PBA/PMMA). The ESI results were found to be in excellent agreement with 1H NMR spectroscopy for PBA/PVAc and PBA/PMMA copolymers whereas there was more divergence in the case of PMMA/PVAc. In the case of PBA/PMMA copolymers similar distributions of products were observed by ESI-MS and MALDI-MS; two major product classes were observed differing by their end-groups. One class has hydrogen and dodecylthio end groups while in the other the dodecylthio has been replaced by alpha-cyanoisopropyl from the initiator. The relative abundance of these distributions as a function of copolymer conversion for a series of reaction conditions was investigated by both ESI and MALDI. The collision-induced dissociation (CID) mass spectra for a variety of chain lengths of four ionized polymer samples have been quantified according to their observed total relative fragment ion abundances. The CID mass spectra of oligomers of ionized PMMA with three different types of end groups and polystyrene, PS, were obtained at fixed centre-of-mass collision energies and collision numbers. For the PMMA polymers, the total fragment ion abundance increases with increasing chain length, consistent with an increase in internal energy deposition with size of the ion. A discontinuity in the increase in total fragment ion abundance appears to correspond to a change in conformation of the polymer ions from linear (at short chain lengths) to cyclic (at long chain lengths). Ionized PS does not exhibit this change in conformation as all chain lengths show compact structures and accordingly the total fragment ion abundance does not change with increasing chain length. Protonation of polymers in the gas-phase was achieved by the dissociation of proton-bound complexes of the oligomers with small peptides and amino acids. Protonated PMMA and PBA oligomers were shown to fragment in unique pathways that involved losses of neutral molecules from their side chains, until all that is left is a hydrocarbon backbone. For PMMA the neutrals were primarily CO and methanol, while for PBA butyl ether is lost. We also explored the threshold fragmentation of the proton-bound complexes as a function of the amino acid used (and its proton affinity) which allowed a kinetic bracketing of the oligomer PA as a function of chain length. The results were consistent with a change from bidentate to tridentate binding of the proton with increasing length of the PMMA oligomers.

The unimolecular dissociation reactions of ionized methylhydrazine, CH 3NHNH2+·, 1,1-dimethylhydrazine, (CH 3)2NNH2+·, and tetramethylhydrazine, (CH3)2NN(CH3)2 +·, were investigated using tandem mass spectrometry, threshold photoelectron photoion coincidence spectroscopy and variational transition state theory. The modelled thresholds for the ion dissociation yielded new thermochemistry for these N2-containing ions. The low energy dissociation pathways of the methyl-substituted hydrazine radical cations correspond to the losses of a hydrogen atom, a methyl radical and/or a rearrangement process leading to the loss of methane. The three fragment ions of MH+· are ion m/z 45, CH 2NHNH2+, ion m/z 31, NHNH 2+, and ion m/z 30, N2H 2+·. The two fragment ions of DMH+· are ion m/z 59, (CH3)(CH 2)NNH2+, and ion m/z 45, CH3N=NH2+. The two fragment ions of TMH +· are ion m/z 73, (CH3)2 NNCH3+, and ion m/z 72, C 3H8N2+·. Product ion structures were determined from MIKE, CID, KER and deuterium exchange experiments performed on a modified VG ZAB mass spectrometer of BEE geometry. The TPEPICO experiments were performed at the Daresbury Laboratory Synchrotron Radiation Source. The breakdown diagrams in the threshold region for each ion were modelled. The potential energy curve of each dissociation reaction was calculated at the B3-LYP/6-31+G(d) level of theory. The molecular configuration corresponding to the minimum sum-of-states was located and used as the transition state in the RRKM calculation of k(E). The k(E) data was convoluted with the internal energy distribution of the ion, the electron transmission function of the electron analyser and the monochromator band-pass width to simulate the experimental breakdown curves. The effects of the entropy of activation on simple dissociation reactions were also investigated. Several transition states were identified with increasing ion internal energy for the methyl radical loss and the hydrogen atom loss channels. The variation in the DeltaS‡ values was found to affect largely the shape of the breakdown curves and the dissociation reactions. Enthalpies of formation of the fragment ions, determined from the theoretical fits of the experimental breakdown curves, were found to be in excellent agreement with the calculated values. The proton affinities of several NN-containing neutral molecules and the N--C and H--C bond dissociation enthalpies were also determined.

This thesis focuses on understanding the interactions of cyanine dyes with DNA, through the use of both experimental and computational techniques. Although cyanine dyes are widely used as nucleic acids stains in fluorescence applications, the nature of the association process is not always clear. The cyanine dye PicoGreenRTM (PG) has proven to be extremely useful in monitoring DNA damage but its structure is proprietary, making it impossible to understand its specific interactions with DNA. However, its structure is known to resemble that of another cyanine dye, thiazole orange (TO). We anticipated that the N-propyl pyridinium derivative of TO (PTO) would also intercalate in DNA and that its extra positive charge, relative to TO, would aid in the association. Our studies have focused on the associations of PG, TO and PTO with DNA. Chapter 3 deals with the association of the dyes with single-stranded DNA homopolymers. The combination of spectroscopic techniques and molecular dynamics (MD) calculations provides a unique understanding of the associations of TO and PTO with single-stranded DNA homopolymers. There is highly specific binding of TO and PTO to poly(dG) and poly(dA), while poly(dC) and poly(dT) bind the dyes very weakly and appear to promote dye aggregation. Due to its proprietary structure, PG could not be studied computationally. However, the experimental spectral results suggest that PG associates differently with single-stranded DNA than do TO and PTO. There are two major findings in Chapter 4. Firstly, the dyes associate strongly with double-stranded DNA, as demonstrated by the experimental spectral results and the MD calculations. Experimental evidence supports not only monomeric dye intercalation in DNA, but also dimeric dye intercalation. The results of the MD simulations suggest that TO and PTO bind to DNA without sequence specificity. The second major finding was that a new type of stable dye/DNA complex is formed when single strands of poly(dA) and poly(dT) are hybridized in the presence of PG or PTO, which cannot be obtained by addition of the dye to poly(dA)·poly(dT). For all three dyes, complete DNA renaturation did not occur during thermal cycling of dye/double-stranded calf thymus DNA. These results suggest that intercalating cyanine dyes can interfere with DNA hybridization to double-stranded DNA. Chapter 5 is concerned with a more practical aspect of the association of cyanine dyes with DNA: using thiazole orange to report UVC-induced DNA damage. A variety of spectroscopic techniques, as well as agarose gel electrophoresis, were examined for their ability to detect UVC-induced DNA damage. The most sensitive methodology of all of those tested was fluorescence spectroscopy using TO. All of the spectroscopic techniques involving TO suggest that TO intercalation is susceptible to UVC-induced DNA damage. The computational studies suggest that the presence of cyclobutadipyrimidines, and not 8-oxo-2'-deoxyguanosine, is a factor in the experimentally observed reduction in dye intercalation. These studies have been a proof-of-concept, to demonstrate the usefulness of this fluorescence technique in detecting high levels of DNA damage, comparable to those used in food irradiation.

Solvated transition metal solvent cluster ions were generated by electrospray ionization and their spectroscopy and photo dissociation dynamics have been studied by laser photofragment spectroscopy. Solvation of Co 2+ by water and methanol has been examined by studying Co2+ (H2O)n and Co2+(CH3OH) n (n = 4–7). An electrospray ionization reflectron time-of-flight mass spectrometer (ESI-RTOFMS) was developed to produce gas phase transition metal solvent cluster ions. This system couples with a Nd:YAG pumped dye laser to produce a unique mass spectrometry-laser photodissociation system. This instrument was compared to ESI-mass spectrometers in the literature. It shows a mass resolution (m/Δm) of 250 and a detection limit of 480 pmol. The instrument compares favorably to existing analytical instrumentation and several improvements are suggested. Hydrated cluster ions, Co2+(H2O)n (with n = 4–7), have been generated by electrospray ionization and studied by laser photofragment spectroscopy. The similarity between the spectrum of gas-phase Co2+(H2O)6 and the absorption spectrum of aqueous cobalt (II) suggests that Co2+(H2 O)6 (aq) is responsible for the room-temperature solution absorption spectrum. The observed photodissociation spectrum of Co 2+(H2O)4 is similar to new bands that appear in aqueous cobalt(II) at high temperatures and have been assigned to Co 2+(H2O)4 (aq) in the literature. The hexahydrate dissociates by loss of one or two water molecules, whereas the heptahydrate dissociates by loss of two or three water molecules. In both cases, loss of two water molecules is the preferred dissociation pathway. The tetrahydrate dissociates either by simple loss of water or by charge separation to form CoOH+(H2O)2 and H3O +, with charge separation being the preferred dissociation channel. Methanolic cluster ions, Co2+(CH3OH)n (with n = 4–7), show behavior similar to that of the hydrated clusters. The gas phase spectra display similar shifts from the solution absorption spectra, but absorb more strongly than the corresponding hydrated clusters. The hexamethanol cluster dissociates via loss of one or two methanol molecules; the heptamethanol cluster dissociates via loss of one, two or three methanols. The tetramethanol cluster primarily dissociates by a charge separation mechanism similar to that observed in the hydrated clusters, forming Co(OCH3) +(CH3OH)2 and H+(CH3OH); dissociation by simple loss of methanol is a minor channel.

Arsenic contamination in groundwater is a worldwide problem. The existing portable field test kits can not provide accurate results when the arsenic concentration is around 10 µg L-1 or lower. This research first was focused on the development and validation of methods in which portable instrumentation, such as electrochemistry instruments or quartz crystal microbalances, could be used to accurately determine arsenic concentrations in water even when the concentration is below 10 µg L-1. A modified anodic stripping voltammetry (ASV) and cathodic stripping voltammetry (CSV) method with measurement at a microarray electrode manufactured by TraceDetect Inc. was developed. When the ASV method with a gold electrode was applied for real water analysis, the detection limit of arsenite was 2.2 µg L-1, and for arsenate was 0.13 µg L-1 . In the CSV method the more commonly used hanging mercury drop electrode was replaced with a mercury film array electrode. Under the optimum condition, this method had a detection limit for arsenite of 0.58 µg L-1 and for arsenate of 2.7 µg L-1. A method for the determination of arsenic using a quartz crystal microbalance was developed in which the crystal surface was modified in situ by dithiolthreitol, an arsenite-selective ingand. The method was applied to real water sample analysis with a limit of 0.6 µg L-1. The second was concerned with an investigation of the kinetics of the reactions that are the basis of several currently available field test kits (as exemplified by the Hach Kit) using inductively coupled plasma mass spectrometry (ICP-MS) with the goal of improving the performance of the test kit. The time for arsine gas reaches to the maximum concentration in the headspace of the vessel was about 60 min without continuous stirring and only 20% of arsenic was absorbed on the test strip. To speed up the arsine generation, continuous stirring condition can be applied. It also made more arsine absorbed on the test strip. The SEM study proves the structure of the darker colored compound. For the lighter colored compounds, the information is not enough to make a conclusion.

The ability to monitor protein aggregation at the molecular level is critical for progress in many areas of life sciences ranging from exploring mechanisms of amyloidosis and etiology of conformational diseases to development of safe and efficient biopharmaceutical products. Despite the spectacular progress in understanding the mechanisms of protein aggregation in recent years, many aspects of the aggregating proteins' behavior remain unclear because of the extreme difficulty in tracking evolution of these notoriously complex and heterogeneous systems. Here, we introduce an electrospray ionization mass spectrometry (ESI MS)-based methodology that allows the early stages of heat-induced aggregation to be studied by monitoring both conformational changes and formation of oligomers as a function of temperature or stress duration. The new approach allows biopolymer behavior (both reversible and irreversible processes) to be monitored in great detail over a wide temperature range. Validation of the methodology is carried out by comparing temperature profiles of model proteins and nucleic acids deduced from MS measurements and differential scanning calorimetry. In order to evaluate the suitability of ESI MS for direct profiling of soluble glycoprotein aggregates, we used heat-stressed human antithrombin, to compare size-exclusion chromatography (SEC) and ESI MS as a means to probe composition of the complex mixture of soluble oligomeric species generated by heat-induced aggregation. Once the appropriate corrections are made, the abundance of the small aggregates derived from ESI MS becomes remarkably close to that calculated based on SEC data, suggesting that ESI MS may be directly applied for at least semi-quantitative characterization of soluble protein aggregates. Application of the methodology to study heat-induced aggregation of human glucocerebrosidase and antithrombin unequivocally links loss of conformational fidelity to formation of soluble oligomers, which serve as precursors to aggregation. Sequential conformational transition of a monoclonal antibody can also be sensitively probed with this method. The ability to make a distinction between various biopolymeric species (based on the differences in their masses) and their conformers (based on the differences in their charge state distributions) allows the temperature-controlled ESI-MS measurements to be carried out in complex systems with very high degree of specificity. This unique feature of the new experimental technique makes it very appealing to the biotech and biopharmaceutical sectors, where the need to engineer/formulate stable biopolymer-based products (e.g., protein drugs) places a premium on the ability to characterize their behavior as a function of temperature with a high degree of precision and accuracy. One of the unique advantages of hydrogen/deuterium exchange mass spectrometry (HDX MS) as a tool to probe protein higher order structure and dynamics is its ability to detect distinct conformational states under certain conditions. When the exchange follows the so-called EX1 or EXX regime, a distinction among various conformers can be made based on the different levels of deuterium incorporation, which manifest themselves in the form of bi- or multi-modal isotopic distributions of protein ions. In this work we exploit this unique advantage of HDX MS and the ability of mass spectrometers to select narrow populations of protein ions to develop a method which allows structure of distinct conformers to be probed at high spatial resolution. Ubiquitin is selected as the model. Validation of the method is carried out by comparing relative magnitude of protection of individual backbone amides deduced from MS and NMR measurements. The two conformers coexisting in the model system exhibit remarkable difference in deuterium incorporation at expected portions of protein sequence. The comparison to reference conformational states of ubiquitin reveals the structural nature of these conformers. These results demonstrate the capability of the top-down HDX MS/MS to specifically capture the conformational features of individual intermediates co-existing at equilibrium.

This dissertation focuses on the mass spectrometric based methods for studying protein-metal binding. Identifying metal-protein interaction is a key step in understanding metal-binding protein structure and function. A phenomenon associated with gas phase dissociation behavior of metal-peptide complexes has been investigated. A positive correlation was found between the number of strong coordination groups in the peptide sequence and the degree of c and z ion formation after electron transfer dissociation of the peptide-metal complexes. Establishing thermochemical cycle enables a theoretical understanding of the process. A new mass spectrometric method has been developed to identify Zn-bound His residues in Zn-metalloproteins relies on variations in the hydrogen deuterium exchange of the C2 hydrogen of His side chains. We show that this approach can be used to study the Zn-bound His residue in human &beta-2-microglobulin; (&beta2m;), a monomeric protein that has been shown to aggregate into amyloid fibrils in dialysis patients leading to dialysis-related amyloidosis. The different effect of three divalent transition metals including Cu(II), Ni(II) and Zn(II) on &beta2m; oligomerization and fibril formation under physiological conditions is described. We found that Cu(II) can induced &beta2m; oligomerization and amyloidosis. In contrast, no oligomeric species can be formed with Ni(II), and only oligomers can be formed with Zn(II). A combination of metal catalyzed oxidation (MCO)-MS, hydrogen deuterium exchange (HDX)-MS and other spectroscopic techniques is utilized to obtain insights into the mechanism of Cu(II)-induced &beta2m; amyloidosis. We elucidate the different ways that these metals bind &beta2m;, thereby identifying key features of the &beta2m-Cu;(II) interaction that are essential for enabling this protein to form amyloid fibrils.

Transition metals play an important role in marine environments. They can be both essential and toxic for marine organisms. These metals are complexed by organic ligands, and their complexes are present at trace levels and in complicated mixtures making their analysis difficult. For this reason, sources and chemical compositions of these species remain unknown. This dissertation is centered on studying the dissociation dynamics of metal complexes in order to use mass spectrometry to get structural information for metal complexes at low concentration levels. This work describes the study of the collision-induced dissociation (CID) patterns and energetics exhibited by different metal complexes in a quadrupole ion trap mass spectrometer. A variety of model ligands containing different donor atoms were synthesized and complexed with first-row transition metals. It was found that differences in the electronic structure of the metal ion, the coordination number of the complex, and the nature of the donor groups bound to the metal are reflected in the types of product ions observed in CID spectra. Upon dissociation, pentacoordinate complexes of Cu and Zn exhibited differences in the preference to remain coordinated to different donor group. For Cu, this preference reflected a balance between the inherent binding strength of the donor group and its flexibility. For Zn complexes, the inherent donor group binding strength and stability of the product ions were more important. The metal electronic structure also impacted the types of dissociation products observed in penta-, and hexacoordinate complexes. Upon CID Cu tends to be reduced. For Zn H2 is the dominant dissociation pathway. Mn, Fe, Co, and Ni Complexes predominantly lose a single binding upon dissociation and sometimes Ni is reduced. Relative dissociation energies were determined by a variable energy CID approach. Metal complexes having metal centers with smaller ionic radii had the lower dissociation energies, except for Cu(II) complexes because its easy reduction. In summary, CID seems to have some promise for helping to distinguish among complexes with different coordinating functional groups, coordination numbers and/or metal centers and thus may some potential for providing coordination structure information for complexes present at trace concentration levels.