Protein ion charge state distributions in Electrospray ionization mass spectrometry (ESI MS) provide information on protein dynamics, as it contains contributions from all protein conformers present in solution. Such ionic contributions often overlap, limiting the amount of useful information that can be extracted from the spectra. This difficulty is overcome by using factor analysis, which allows spectral deconvolution to be carried out and information on individual protein conformers to be extracted. Experiments were carried out by acquiring a series of spectra over a range of near-native and denaturing conditions and then subjecting to singular value decomposition, to determine the number of conformers. Ionic contributions of each conformer to the total signal are then determined using a supervised minimization routine. By monitoring acid and alcohol induced equilibrium intermediates of well-characterized model proteins we can demonstrate that factor analysis correctly predicts the total number of conformations of the model proteins under a variety of conditions. Several factors have also been suggested as determinants of the number of charges displayed by the protein ions in electrospray ionization (ESI) spectra, such as available basic residues, solvent composition, etc. This work demonstrates that protein surface area in solution is the major determinant of the number of charges accommodated by a protein ion in the gas phase. The charged residue model of ESI process predicts that the number of charges (N) accommodated by a globular protein in the gas phase should be related to its surface area (S) as N∼S3/4. We analyzed a set of proteins ranging from 5 kDa insulin to 0.5 MDa, ferritin under native conditions. The average charge of each protein was plotted as a function of its surface area (calculated based on the available crystal structures), giving an apparent correlation of N∼S0.68. Effects of gas phase chemistry on the charge state distribution was investigated. It is seen that using a stronger base as a electrolyte component resulted in a noticeable decrease of the average charge, without affecting the correlation N∼S 0.68. At high pH charge state distribution is shifted to lower charges, attributed to the free base in solution.

Monitoring hydrogen-deuterium exchange of proteins can yield a wealth of information about not only the native state, but also not native states of proteins. These non-native states have important biological roles including protein folding intermediates as well as ligand binding/delivery and protein-protein interactions. Structural and dynamic properties of a partially folded conformation (A-state) of ubiquitin are studied using amide hydrogen exchange in solution (HDX) and mass spectrometric detection. A clear distinction between the native state of the protein and the A-state can be made when HDX is carried out in a semicorrelated regime. Furthermore, combination of HDX and protein ion fragmentation in the gas phase by means of collision-induced dissociation (CAD)] is used to evaluate the conformational stability of various protein segments specifically in the molten globular state. Chain flexibility appears to be distributed very unevenly in this non-native conformation. This study also demonstrates the power of mass spectrometry as a tool in providing conformer-specific information about the structure and dynamics of both native and non-native protein states coexisting in solution under equilibrium. This dissertation has been broken down into several subsections. First, we evaluate electrospray ionization amide hydrogen exchange collision assisted dissociation mass spectrometry's (ESI HDX CAD MS) methodology to better understand the determinants of hydrogen scrambling in the gas phase, which can be used to probe non-native states of proteins. Secondly we examined the structure of a molten globule of ubiquitin using HDX CAD MS under mildly denaturing conditions and compare this with the proposed NMR and crystal structures of the A-state and native state, respectively. Lastly, we have conducted studies include ESI MS studies of non-covalent interactions between cellular retinoic acid binding protein II (CRABP II) and the ligand-binding domain (LBD) of retinoic acid receptor (RAR) as it pertains to delivery of retinoic acid (RA). The backbone dynamics of CRABP II will be investigated in order better understand the ligand binding and delivery properties of CRABP II and how they differ from CRABP I.

The selectivity and sensitivity offered by the combination of liquid chromatography (LC) and mass spectrometry (MS) make it a powerful tool for studying the chemistry of compounds that are of environmental and biological importance. This dissertation focuses on developing LC-MS methods to study metal-interacting molecules in marine and biological systems. In the studies of marine systems, organic ligands that bind Cu(II) in estuarine waters and molecules that are produced by the archaeon Pyrobaculum aerophilum to reduce Fe(III) to Fe(II) have been investigated. Two groups of Cu(II)-binding ligands in the Chesapeake Bay have been isolated by immobilized-metal affinity chromatography (IMAC). Reversed-phase liquid chromatography (RPLC) analyses indicate that both groups of ligands are hydrophilic in nature. Further MS analyses indicate that at least one of the ligands is made up of sulfur- and nitrogen-containing functional groups. LC-MS has also been used to isolate and characterize molecules that are produced by the archaeon Pyrobaculum aerophilum to reduce Fe(III) to Fe(II). Further characterization of these compounds by UV-Vis spectroscopy, NMR spectroscopy, and tandem MS (i.e. MS/MS) indicates that at least one of these compounds contains amide and quinone-like groups. In the studies of biological systems, the gentle nature of electrospray ionization (ESI)-MS has been used to study metal-protein and protein-protein complexes of β-2-microglobulin (β2m), which is the protein component of the amyloid fibers that cause dialysis-related amyloidosis. MS, size-exclusion chromatography (SEC), and dynamic light scattering (DLS) analyses indicate that in the presence of Cu(II) β2m forms amyloid fibers by the building up of dimeric units. Furthermore, MS, SEC, and DLS data suggest that the hexamer is the nucleus that is required for the formation of the amyloid fibers. Taken as a whole, MS, SEC, DLS, and X-ray fluorescence data also suggest that Cu(II) is necessary to reach the hexameric state, but Cu(II) is released upon formation of the mature fibers. Finally, ESI-MS has been used to estimate the surface areas of β2m oligomers. Our surface area measurements suggest that the hexamer has a more compact structure than the dimer or tetramer, suggesting that Cu is released by the tetramer upon formation of the hexamer.

A method was developed for preconcentration, separation, and determination of arsenic species [As(III), As(V), DMA, and MMA] in water samples by flow injection solid phase extraction hydride generation atomic absorption spectrometry. Competing anion interferences were minimized by employing two anion-exchange columns in series. The method was successfully applied for the determination of trace concentrations in various water samples spiked with a mixture of arsenic species. A new method was developed for the determination of ultra-trace amounts of As(III) and As(V) by flow injection solid phase extraction hydride generation atomic absorption spectrometry with tetrahydroborate immobilized on an anion-exchange substrate. The method offers several advantages including simplicity, sensitivity improvement, arsenic speciation, cost reduction and less waste generation compared to the conventional hydride generation AAS method. The method was successfully applied to the determination of As(III) and As(V) in different kinds of environmental water samples including well water, lake water, tap water, spring water, and sea water. A new method was developed for the determination of As(III), As(V), DMA and MMA by high performance liquid chromatography hydride generation inductively coupled plasma optical emission spectrometry. The new multimode sample introduction system (MSIS) was employed instead of a conventional gas-liquid separator. The separation was achieved with good resolution and detection limits by using an anion-exchange column, either an IonPac AS10 or an Alltech Anion HC column. The extraction of arsenic species from soils was significantly improved with a sequential extraction procedure. Arsenic species extracted were successfully separated and determined by high performance liquid chromatography hydride generation inductively coupled plasma optical emission spectrometry with the MSIS. No significant changes in the arsenic species were observed during the extraction. A method was developed for improving the detection of the Hach test kits. Solid phase extraction was used for the preconcentration of arsenic in water samples prior to testing by the Hach kit. The color intensity for all concentrations including those close to 10 μg l-1 was increased. The method was successfully applied to environmental water samples spiked with low arsenic concentrations.

The instrumental limit of detection (LOD) lies at the heart of modern analytical chemistry, yet there is still much debate as to its role and reliability as a figure of merit. There are a number of possible ways to define the LOD, and in the present work four such sample test statistics were defined and their probability density functions (PDFs) were derived. The PDFs were easily evaluated via numerical integration and were used to obtain expectation values, population precisions, and confidence intervals. Monte Carlo simulation methods were used to prepare normalized histograms of one million LOD variates each, for homoscedastic linear calibration curve systems, and these were found to be in excellent agreement with the numerically obtained PDFs and associated statistics. With the realization that LODs are random variates governed by their PDFs it becomes evident that the quotient of two LODs is also a random variate governed by its own distribution. One case of a limit of detection definition was taken and the theoretical PDF for the quotient of two LODs was derived. Monte Carlo simulations were again used to generate normalized histograms of one million quotient of LOD variates, and were also in excellent agreement with the numerically evaluated theoretical PDF. In addition to the simulation and theoretical approaches, a real chemical measurement system was designed in an effort to collect large data sets that could be used to obtain experimental LODs. The data collected on the measurement system was carefully analyzed and, after a randomization algorithm, was deemed appropriate to generate a normalized histograms of quotients of LOD (in some cases up to 40,000 points). It was found that the distribution of values obtained from the real system almost perfectly matched that of the derived theory and Monte Carlo simulation providing a sound argument for the conclusions we have drawn. The software used in all aspects of the work is available with full commented source code.

Methods for the determination of trace elements in environmental and biological samples were developed and optimized. Parameters affecting sample introduction efficiency, matrix effects, sample throughput and tolerance to interfering species were evaluated for methods based on plasma spectrochemistry and electrochemistry. A method was developed for the determination of copper in the presence of β-2-microglobulin. A mass balance of copper was performed at the end of in vitro incubations which required method development to quantify copper in the liquid and solid portions of the incubation solutions, along with copper adsorbed and mass transferred into the walls of the incubation vessel. A flow injection method was developed to accommodate the small sample volume and high dissolved solids content to allow for the determination of copper by ICP-MS. A commercial introduction system was evaluated for the determination of trace elements in drinking waters and wastewaters by ICP-MS and ICP-OES. Parameters including increased throughput, reduced memory effects, increased stability and lower reagent consumption were evaluated as the system was successfully applied to U.S. EPA Methods 200.8 and 200.7, respectively. Particular attention was paid to the retention of mercury and sodium and long term stability during the analysis of samples containing high total dissolved salts. Results obtained with the new introduction system were compliant with EPA Methods 200.8 and 200.7 while increasing sample throughput two- or three-fold and significantly reducing memory effects. A method was developed for the enhancement of signals for trace elemental analysis by DRC-ICP-MS. Signal enhancement was evaluated as instrumental and reaction gas parameters were optimized. Background noise levels remained relatively unchanged as signal intensities were improved, resulting in improved signal to noise ratios. The developed method was successfully applied to the determination of gold in synthetic geological samples. A method was developed for the preconcentration and speciation of arsenic by anodic stripping voltammetry using ion exchange resins. Ion exchange and instrument parameters were optimized. The developed method was successfully applied to the determination of arsenic(III) and arsenic(V) in waters without interference from problematic species such as Cu(II). Suitable detection limits were obtained and the method was validated using a certified reference material.

Methods for the simultaneous determination and speciation of arsenic, selenium and antimony in complex matrices involving inductively coupled plasma optical emission spectrometry (ICP-OES), and inductively coupled plasma mass spectrometry (ICP-MS) coupled with high performance liquid chromatography (HPLC) were developed and successfully applied to the analyses of various types of environmental and biological samples. A flexible internal standardization procedure for ICP-OES has been developed that extends the choices of possible internal standards and has the potential for greater accuracy than traditional internal standardization based on one standard. This method is based upon the visualization by principal component analysis of the similarity of matrix-induced behavior among emission lines. For a given analytical line, more choices of internal standards are provided from composite spectral lines created by combining real spectral lines in such a way as to integrate their characteristic behaviors. The method was applied with satisfactory results to the determination of As, Sb and Se in solutions which modeled plant and food sample digests to compensate for the matrix effect. A method for the simultaneous determination of As, Sb and Se by the multimode sample introduction system coupled to ICP-OES has been developed. Reaction mechanisms for sample treatment and hydride generation are proposed to account for some unusual observations. In order to study urinary arsenic and selenium metabolism and interactions in the human body, a procedure for the simultaneous determination of six Se species and six As species in human urine by HPLC-ICP-MS has been developed. Positive associations between As intake and urinary Se metabolites, Se intake and urinary As metabolites were found. As and Se interactions may increase the mobility of both species in human tissue or blood, and as a result, more As and Se may be excreted. The competition for methyl groups between As and Se species changes the distribution of As and Se metabolites in human urine. The excretion of methylated species are delayed. The formation of monomethylated species is enhanced while that of multimethylated species is inhibited when both As and Se are ingested.

The precursor of open chain Reissert analogues was prepared by reaction of mandelonitrile with aniline to form 2-anilino-2-phenylacetonitrile. A range of open chain Reissert compounds was made by reacting the acetonitrile with p-nitrobenzoyl chloride, p-methylbenzoyl chloride, and p-methoxybenzoyl chloride. Each of the Reissert compounds was treated with fluoroboric acid to form the munchnone iminium salt. Each was reacted with ethyl propiolate and ethyl phenylpropiolate to yeild highly substituted pyrroles. The ratio of the regioisomeric products of each of the reactions was established by HPLC. Identification of the regioisomers was accomplished by alternate syntheses, and by 300 MHz ('1)H-NMR analyses. Based on the regiochemical data gathered, 1,3 dipolar cycloaddition reactions of munchnone iminium salts with substituted acetylenes are highly regioselective. Regioisomeric product ratios indicate that merostabilization has a greater effect on the regiochemistry of the reaction than Linnett stabilization, which in turn is stronger than benzylic stabilization for the reactions of munchnone iminium salts with substituted acetylenes.

Mass spectrometry-based proteomics has become an important and versatile tool in analytical chemistry, making sense of complex biological samples and shedding light on the intricate proteomes of living organisms. Bottom-up proteomics studies are used to elucidate the changes in gene expression of bacteriophage T7 over the course of infection of Escherichia coli. E. coli cultures were infected with T7, sampled over time, and proteins were isolated and enzymatically digested. Nanoflow liquid chromatography combined with tandem mass spectrometry was used to detect proteolytic peptides and identify host and phage proteins. Generally, phage proteins were detected on a time scale fitting the established lytic cycle for T7 phage, confirming the effectiveness of infection monitoring by mass spectrometry-based proteomics studies. Continued development of the experimental method sought to increase detection of proteolytic peptides and identify phage and host proteins to a higher level of confidence, and lead to the implementation of 1D SDS-PAGE as a fractionation method to reduce sample complexity and increase method sensitivity.

The goal of this dissertation is to develop applications for metal selective gas chromatographic detection by microwave-induced plasma atomic emission spectroscopy. A new commercial GC-AED system is used in the examination and analysis of a wide variety of metal-containing compounds. The performance of the GC-AED for selective detection of model compounds containing aluminum, gallium, chromium, vanadium, cobalt, nickel, palladium and copper is evaluated. Figures-of-merit are reported for these compounds. For aluminum, gallium, chromium, vanadium and palladium, new recipes are developed for the AED concerning digital filters for continuous background correction and experimental parameters such as emission wavelength, helium flow rate and reagent gases, as well as discharge tube materials. The separations of the diastereoisomers of VO(pnTFA$\sb2$) are demonstrated by both capillary GC and HPLC. GC-AED proved valuable in the study of gas phase diastereoisomerism of the tetradentate oxovanadium Schiff base chelates. Picogram detection limits for vanadium by GC-AED facilitated study of kinetic behavior at low concentrations. Multielement detection for metal and nonmetals in the chelates provided identification of eluted peaks. HPLC coupled with DCP and PDA detection assisted the study of the isomerization in solutions. Methods are developed for the characterization of chromium $\beta$-diketone polymers by GC-AED and GC-MS following pyrolysis. Thermal stability is reported for a series of chromium and cobalt chelates and their polymers. Element specific detection proved to be superior in screening metal-containing pyrolysates and simplifying the interpretation of other fragments. High temperature GC-AED is developed for the analysis of vanadyl, nickel and iron porphyrins in crude oils. Limits of detection, precision and inter-element selectivity between vanadium, nickel and iron are reported. The metal elution profiles are characteristic for different oils and compatible for fingerprinting the oils from different fields. The method is also advantageous in the study of decomposition of petroleum metal species. Quantification of the metal profiles indicates the contents of the distillable metals to be generally lower than those of total metals determined by the direct spectroscopic methods. A study on the distribution of metalloporphyrins in crude oils is also carried out by gel permeation chromatography. A correlation between total metal, metalloporphyrin and total distillable metal contents is addressed in this study. Sample pretreatment procedures to remove heavy oil matrices prior to the GC-AED analysis are investigated. Solvent extraction in combination with solid phase extraction is used to extract volatile metal species favorably. GPC proved to be even more effective and reproducible in the resolution of volatile metal species into clean and defined fractions.