Increasing speciation demands in clinical chemistry, toxicology and nutrition have made the determination of the total elements in a sample inadequate; the amount of an element and the chemical forms in which it is present need to be known. Inductively coupled plasma mass spectrometry (ICP-MS) was used after high performance liquid chromatographic (HPLC) separation, as was electrospray ionization mass spectrometry (ESI-MS) for the characterization of selenium compounds. Consumption of selenium enriched plants or yeast-based nutritional supplements has been reported to provide anticarcinogenic benefits which are selenium compound dependent. Separation and identification of these selenium compounds is critical to understand the activity. Plants and yeast convert inorganic selenium in the soil or growth media into organoselenium compounds, probably following a route similar to the sulfur assimilatory pathway. Non-volatile selenium compounds produced include selenoamino acids, some of which have shown anticarcinogenic activity. A series of ion-pair chromatographic methods with ICP-MS and ESI-MS detection for the separation, qualitative and quantitative determination of non-volatile selenium compounds extracted from different samples has been developed. The method also provides the capability of determining the presence of selenoxides. Results of total selenium determination and speciation of selenium enriched yeast-based nutritional supplements, selenium-enriched Allium vegetables and phytoremediation samples are presented. More than 75% of the total eluting compounds were identified from retention time data and standard spiking experiments, and between 60 and 85% of compounds were identified by MS. Mass spectral behavior of selenoamino acids, using electrospray and ion trap technology with direct infusion and liquid chromatographic sample introduction, is also reported.

Retinoic acid (RA) is the most potent metabolite of vitamin A and is utilized in signaling pathways that control many cellular events. Cellular retinoic acid binding proteins I and II are carriers of RA inside the cell and CRABPII transports RA to the nucleus where it binds to the retinoic acid receptors (RARs) and can then exert its effect on the transcription of genes. CRABPI is thought to bind excess RA in the cell cytoplasm to protect it from the oxidizing effect of RA. The interaction between CRABPs and RARs is very transient and such a complex has never been observed. Entry and exit of RA in CRABP occurs through fluctuation of the protein backbone through the portal region. Based on this and the crystal structures of CRABPs, a mutant containing a disulfide bond in the portal region it the holo-form of the protein was designed. This mutant effectively “locks” RA inside its binding cavity in the presence of lipid vesicles, confirming that ligand movement in and out of CRABP necessitates backbone fluctuation. Using a combination of Bioaffinity chromatography and hydrogen deuterium exchange mass spectrometry, the mechanism of transfer of RA during the CRABP/RA/RAR interaction was studied. Holo-CRABP was more protected than the apo-form and the presence of RAR did not result in a change in the protection level of apo-CRABP. However, the exchange kinetics of holo-CRABP in the presence of RAR were different from that of apo- or holo-CRABP alone. The CRABP mutants containing a disulfide bond in the portal region have an initial exchange rate that is higher than for the wild type CRABP. The complexes between those holo-mutants and RAR have a longer half-life than the wild type CRABP. The accelerated hydrogen deuterium exchange kinetics of the holomutant in the presence of RAR could therefore not be the result of faster ligand transfer from CRABP to RAR. These results were observed with both CRABPI and CRABPII. This led to a model in which during RA transfer from CRABP to RAR, the backbone of CRABP is destabilized through its interaction with RAR.

The role of metallo-proteins in the body and their speciation is of increasing interest in clinical diagnostics because it is well recognized that their properties, function, and regulation in metabolic processes are highly species dependent. Therefore, their use as health markers for in vitro diagnostic, medical care and nutrition is directly related to their biological role. Selenium-containing proteins are important examples of speciation related health-indicators. In this work, significant progress has been made on the development of analytical methods for the speciation of selenoproteins in serum. A complete two-dimensional chromatographic separation of the different species of selenium-containing proteins of clinical interest including albumin, glutathione peroxidase and selenoprotein P is presented. Metal (Co 2+) chelate affinity high performance liquid chromatography (HPLC) was used to extract the proteins from serum, replacing the traditional low-pressure chromatography. Reversed phase chromatography was used for desalting and further separating selenoprotein fractions. The selenoproteins were identified by a combination of UV detection at 280 nm, selenium measurement by DRC-ICP-MS, and peptide mapping and molecular mass determination by MALDI-MS. Quantification of selenium in the selenium-containing protein species was accomplished using HPLC-UV-DRC-ICP-MS analysis employing 82Se stable isotope dilution. The sample was partially digested on line after separation, with nitric acid, using a novel in house designed interface (IRIS Digester-Evaporator, for which a patent has been applied). This interface reduces nitric acid and organic solvent from the liquid flow, and permits direct connection to the DRC-ICP-MS sample introduction system. It is a choice for HPLC-ICP-MS application or for faster on-line sample preparation for ICP and FAAS systems. The 2D chromatography, particularly the IMAC was the major source of variability in the overall method.

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.

A thorough understanding of the chemical and physical properties of small molecules involves spectroscopy, thermodynamics, kinetics and dynamics of the system. This thesis highlights the use of photochemistry to measure these properties for Au+(C2H4), Pt+ (C2H4) and ethylbenzene cation as well as for deposition of zeolite films. Chapter 1 highlights the principles behind photodissociation and the information that can be obtained from spectroscopy. Also discussed is what the appearance of the photodissociation spectrum and time-of-flight profile implies about the molecular dynamics and kinetics. Chapter 2 examines gas-phase photodissociation of the classic π complexes Au+(C2H4) and Pt+(C 2H4). Spectroscopic onsets provide upper limits to the metal-ligand bond strengths of 344 kJmol−1 and 230 kJmol−1 for Au+(C2H4) and Pt +(C2H4) respectively. The spectrum of Au +(C2H4) features an extended progression in the metal-ligand stretch with a frequency of 176 cm−1 that drops to 160 cm−1 in Au+(C 2D4). Hybrid density functional theory (DFT) calculations and TD-DFT calculations are used to explore the structures, bonding and electronic spectroscopy of the two complexes. The photodissociation pathways and kinetics of the ethylbenzene radical cation are investigated in Chapter 3. The energy dependence of the various pathways are investigated. The most abundant dissociation channel is C 7H7+ + CH3 at all wavelengths, but C6H4+ + C2H6 and C6H6+ + C2H4 are also important in the near-UV. The C6H4+ + C2H6 pathway is especially interesting as it exhibits a significant peak broadening with ∼0.6 eV kinetic energy release. Later studies using vibrationally cold ions examined the dissociation rate, k(E), at various photon energies. Simulations of the time of flight profile show that the k(E) increases from 0.97 × 106 s−1 at 2.38 eV to 2.6 × 106 s −1 at 2.67 eV internal energy. An alternate application of molecule-light interaction to surface science is explored in Chapter 4. Here, the technique of pulsed laser deposition has been employed in efforts to improve thin film growth for ETS-4 molecular sieves. Characterization using X-Ray Diffraction and Transmission Electron Microscopy confirm that the zeolite structure is unaltered during the deposition process. Recommendations for further studies are discussed in Chapter 5.

Otolith microchemistry can be very useful in identifying fish populations and reconstructing fish movements. Recent attempts have been made to evaluate otoliths as proxies of ambient transition metal levels, but findings have been inconsistent, varying by metal, species, and experimental method. Some of the difficulty with obtaining definitive answers stems from an incomplete understanding of the biological controls governing transition metal speciation in otoliths. Metals may be incorporated into the calcium carbonate crystal structure, trapped in crystalline interstitial spaces, or associated with the protein matrix. Metal binding to the protein phase may be inferred from its structural and biochemical properties but has not been observed previously. Inherent difficulties with the extraction of metal-binding proteins in their native state from the calcium carbonate phase make them extraordinarily difficult to measure. A method has been developed that facilitates the extraction of otolith proteins without total disruption of transition metal-binding. Chelating agents such as EDTA, used in otolith decalcification, can demetallate the proteins if the demineralization is allowed to reach equilibrium; however, if the reaction is halted prior to equilibration, intact metal-protein complexes can be obtained. Using such an approach, we have established that between 70% and 100% of Cu and 40% to 60% of Zn found in whole otoliths are associated with the soluble portion of the protein matrix. Mn was not observed to be associated with the protein, indicating that it is either weakly bound or that no protein-bound Mn is present. Our results, combined with knowledge of the biological control of these metals, suggest that otoliths are unlikely to be reliable indicators of Cu and Zn exposure, but may provide a useful and unique insight into fish growth and physiological development. We have also partially characterized the protein matrix of the otolith itself, and developed analytical methods which will facilitate further study into the nature of this complex system. We have observed multiple water-soluble proteins over a large mass range, many of which are glycosylated. This work will aid in furthering the understanding of otolith development, which is essential if trace metal signatures in otoliths are to serve as bioindicators.

Sulfur and selenium participate in a variety of essential biological functions. The investigation of two non-heme iron dioxygenases with sulfur-containing substrates is reported along with studies of seleno-compounds by gas chromatography with atomic emission detection (GC-AED). Cysteine dioxygenase (CDO) catalyzes the oxidation of cysteine to cysteine sulfinic acid, which is the first major step in cysteine catabolism in mammalian tissues. Rat liver CDO was cloned and expressed in E. coli as a 26.8 kDa fusion protein bearing a poly-histidine tag. Kinetics studies revealed a Km value of 2.5 ± 0.4 mM at pH 7.5 and 37 °C, with no requirement for secondary proteins or cofactors. Fe was demonstrated to be the only metal that is essential for activity. Inhibition studies with cysteine analogs along with the use of x-ray absorption spectroscopy (XAS) allowed for the characterization of the active site. Acireductone dioxygenases (ARDs) are enzymes involved in the methionine recycle pathway. Klebsiella produces two ARD enzymes that share a common polypeptide sequence and differ only in the metal ion present. In the presence of Fe-containing ARD (ARD'), reaction of acireductone with dioxygen produces formate and the ketoacid precursor of methionine. In the presence of the Ni-bound form (ARD) the same substrate produces formate, methylthiopropionate and CO, an off-pathway shunt. XAS study of the structure of the catalytic Fe center in resting state enzyme shows a six coordinate Fe site composed of N/O-donor ligands including 3-4 histidine residues. The substrate binds to the Fe center in a bidentate fashion by displacing two ligands, at least one of which is a histidine ligand. Cancer prevention attributed to the properties of selenium is widely recognized, but the mechanism of tumor inhibition by this element is still not known. The determination of Se-metabolites and the understanding of their fate are indispensable. Liver extracts from rats administered with selenized yeast were examined by GC-AED. Organoseleno compounds have been observed, including selenomethionine, and possibly the newly-discovered S-(methylseleno)-cysteine molecule. Aromatic Se-compounds have been recognized as antitumorigenic alternatives with lower toxicity. Gas chromatographic behavior of several aryl diselenide derivatives were analyzed.

The development of highly selective and very sensitive methods to detect peptides and proteins of interest in complex mixtures remains an important goal in proteomics applications. This dissertation focuses on the use of reverse-micelle forming amphiphilic homopolymers as part of liquid-liquid extraction to selectively extract and concentrate peptides from an aqueous solution into an immiscible organic phase. After extraction, the polymer-peptide mixtures are amenable to direct analysis by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). The charged interiors of the reverse micelles enable oppositely charged peptides to be selectively extracted into the aggregate’s cores via coulombic attraction. Reverse micelles formed by negatively charged carboxylic acid or positively charged quaternary amine groups can be used alone or in sequence to selectively extract and fractionate peptides according to their isoelectric points (pIs). Furthermore, the pI cutoff can be readily tuned by adjusting the extraction pH. The coalescence of polymer-peptide conjugates into hotspots on the MALDI target plate during MALDI-MS analysis results in significant signal enhancement for the enriched peptides, enabling reproducible ion signals at concentrations as low as 10 fM. Interestingly, reverse micelles formed by positively charged polymers with quaternary amine substituents can selectively enrich acidic peptides that are undetectable during regular MALDI-MS analysis. The extraction protocol along with MALDI-MS can also be used for the selective enrichment and detection of low abundance peptide/protein biomarkers in human serum at physiologically relevant concentrations. Overall, the results described in this dissertation reveal that this selective extraction protocol along with MALDI-MS analysis might have a significant impact on protein identification and early stage screening of biomarkers.

A multiplexed method for performing MS/MS on multiple ions simultaneously in a miniature rectilinear ion trap (RIT) mass spectrometer has been developed. This method uses an ion encoding procedure that relies on the mass bias that exists when ions are externally injected into an RIT operated with only a single phase RF applied to one pair of electrodes. The ion injection profile under such conditions ions is Gaussian-like over a wide range of RF amplitudes, or low mass cutoff (LMCO) values, during ion accumulation. We show that this distribution is related to ion m/z, and is likely caused by ions having an optimal range of pseudo-potential well depths for efficient trapping. Based on this observation, precursor ion intensity changes between two different injection LMCO values can be predicted, and these ion intensity changes are found to be carried through to their corresponding product ions, enabling multiplexed MS/MS spectra to be deconvoluted. The gas-phase reactions of a series of coordinatively unsaturated [Ni(L) n]y+ complexes, where L is a nitrogen-containing ligand, with chemical warfare agent (CWA) simulants in a miniature rectilinear ion trap mass spectrometer were investigated as part of a new approach to detect CWA. Results show that the metal complex ions can react with low concentrations of several CWA simulants, including dipropyl sulfide (simulant for mustard gas), acetonitrile (simulant for the nerve agent tabun), and diethyl phosphite (simulant for nerve agents sarin, soman, tabun, and VX), thereby providing a sensitive means of detecting these compounds. The [Ni(L)n] 2+ complexes are found to be particularly reactive with the simulants of mustard gas and tabun, allowing their detection at low parts-per-billion (ppb) levels. These detection limits are well below the median lethal doses for these CWAs, which indicates the applicability of this new approach, and are about two orders of magnitude lower than electron ionization detection limits on the same mass spectrometer. The use of coordinatively unsaturated metal complexes as reagent ions offers the possibility of further tuning the ion-molecule chemistry so that desired compounds can be detected selectively or at even lower concentrations. Mass spectrometry has become a tool for studying noncovalently bound complexes. Specifically, electrospray ionization mass spectrometry (ESI-MS) has found increasing use for the determination of affinity (Ka) or dissociation (Kd) constants. Direct measurement of the equilibrium components by ESI-MS is the most straightforward approach for determining binding equilibrium constants, but this approach is prone to error and has some inherent limitations. Transferring complexes from solution to the gas phase may perturb the equilibrium concentrations and/or different ionization efficiencies may cause the resulting ion signals not to reflect actual solution concentrations. Furthermore, ESI only works under a limited range of solvent conditions (i.e. low ionic strengths), which limits the broad applicability of this approach. We propose an approach based on covalent labeling in the context of metal-catalyzed oxidation (MCO) reactions that, when combined with MS, overcomes such limitations when determining metal-ligand binding constants. The MCO-MS approach will provide concurrent information regarding metal binding site and metal-protein binding affinity. Optimization of the MCO reaction through isotopic mass tags will permit enhanced identification of modified residues. Application of this method to study the affinity and binding interactions of other divalent metals with β2m are likely to provide insight into the specificity of copper for causing β2m amyloid formation.

In the first part of this project a refractive index sensor was designed that will ultimately allow the study of ligand-protein and protein-protein interactions. The setup consists of a flow cell that contains an integrated optics Mach-Zehnder interferometer chip, into which laser fight can be introduced by end- or prism-coupling. The optical parameters of the interferometer were chosen to achieve sensitivities as low as 0.07 ng/cm2. Optimized procedures for the interferometer fabrication, dimensions of the individual sensor parts as well as parameters for the optical alignment are described in great detail. While successful light coupling into the waveguiding channel of the interferometer was achieved, improvements in the light conductivity of the titanium-silica waveguides are still necessary to make this setup work. A second issue of this thesis is the preparation of a polymeric matrix for biosensors, that consists of dextran functionalized with nickel(II)nitrilotriacetic acid (NiNTA-dextran) and the characterization of its binding affinity toward histidine oligopeptides and histidine-tagged proteins by isothermal titration calorimetry (ITC). Enhanced binding affinities of the oligopeptides to NiNTA-dextran relative to NiNTA that are due to polyvalent interactions between the NiNTA-moieties on the modified dextran and neighboring histidine groups in the peptides were found. An increase in the strength of the interaction between NiNTA-dextran and the oligohistidines was observed as the number of histidine residues in the oligopeptide were increased. Proteins with at least four neighboring histidine groups are expected to be particularly suited for the generation of stable affinity matrices out of NiNTA-dextran. A decrease in the stability of the NiNTA-dextran matrices that are loaded with histidine-tagged proteins by buffering reagents was demonstrated, underscoring the importance of choosing an appropriate buffer system in biosensor applications. Finally a model for the structure of complexes between Ni-NTA and oligohistidine residues is proposed based on the stoichiometry of binding observed by ITC.