Three Miller index surfaces of copper, Cu(111), Cu(211), and Cu(643) were evaluated for spontaneous carbon dioxide dissociation. DFT (Density Functional Theory) was used to characterize the initial and final adsorption states and Climbing Image Nudged Elastic Band (cNEB) calculations were used to identify the dissociation transition sites. A simple kinetic model was formulated and used to quantitatively compare the three surfaces and determine which facilitated CO₂ dissociation most readily.

Thesis (Ph.D.)--University of Central Florida, 2009. / Adviser: Viatcheslav Kokoouline. Includes bibliographical references (p. 138-149).

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

This thesis presents a modeling program to obtain equilibrium information for a fluorescent system. Determining accurate dissociation constants for equilibrium processes involving a fluorescent mechanism can prove to be quite challenging. Typically, titration curves and non-linear least squares fitting of the data using computer programs are employed to obtain such constants. However, these approaches only consider the total fluorescence signal and often ignore other energy transfer processes within the system. The current model considers the impact on fluorescence from equilibrium binding (viz., metal-ligand, ligand-substrate, etc.), quenching and resonance energy transfer. This model should provide more accurate binding constants as well as insights into other photonic processes. The equations developed for this model are discussed and are fit to experimental data from titrimetric experiments. Since the experimental data are generally in excess of the number of parameters that are needed to define the system, fitting is operated in an overdetermined mode and employs error minimization (either absolute or relative) to define goodness of fit. Examples of how changes in certain parameters affect the shape of the titrimetric curve are also presented. The detection of metal ions is very important, causing a need for the development of a metal ion sensor that provides selectivity, sensitivity, real-time in situ monitoring, and a flexible design. In order to be able to perform in situ monitoring of trace metal ions, FRET-pair labeled peptides were attached to a Tentagel[trademark] resin surface. After soaking in nonmetal and metal solutions (pH = 7.5), the resin beads gave an enhanced response in the presence of Hg²⁺ and Zn²⁺. Using a t-test, the signals of the beads that were soaked in a solution of each of these metal ions (and that of Cd²⁺) were determined to be significantly different from beads soaked in a solution without metal. However, the standard deviation between a set the beads was too large in order to differentiate a bead that was soaked in nonmetal solution versus one soaked in a metal containing solution. / text

Photodissociation methods have been explored for structural analysis of peptides and oligosaccharides. Ultraviolet photodissociation (UVPD) was applied to carboxylated derivatized peptides and reducing end derivatized oligosaccharides which offer selective dissociation and specific fragmentation pathways in comparison to CID. Upon UVPD of the modified peptides at carboxylate comprised of reduced y ions and increased immonium ions. The derivatized oligosaccharides via reductive amination and hydrazide conjugation can undergo highly efficient 355 nm UVPD and offer different fragmentation pathways. Both derivatization methods upon UVPD yielded [superscript 0,2] A-type ions, however reductive amination and hydrazide conjugation produced dominant [superscript 0,1] A and [superscript 2,4] A-type ions, respectively. Ultraviolet photodissociation at 193 nm (ArF laser, 6.4 eV / photon) has been applied to sialylated oligosaccharides and glycans which were analyzed in negative mode due to their acidic condition. Primarily, UVPD provides a greater array of fragment ions including cross-ring cleavages and dual cleavage internal ions in comparison to CID. In addition, the UVPD generates unique fragment ions which arise from site-specific cleavage of the trial substituent of the sialic acid residue. UVPD of doubly deprotonated sialylated oligosaccharides produced mostly singly deprotonated fragment ions, whereas the product ions in the CID spectra were overwhelmingly doubly charged ions, an outcome attributed to the more extensive cleavages of sialic acid residue upon UVPD. Although electron transfer dissociation (ETD) has shown superior capabilities for the characterization of post-translational modifications of peptides due to its non-eragodic property, ETD has intrinsic drawback arising from its significant dependence on the charge state of the selected precursor ion. Precursor ions in low charge states tend to undergo charge reduction, often preferentially relative to production of the informative cand z-type ions. In order to increase charge states of peptides and ETD efficiencies, peptides were derivatized at their carboxylate groups via attachment of amine with fixed charge or hydrophobic group. The carboxylate-derivatized peptides exhibited higher ETD efficiencies relative to underivatized peptides along with greater numbers of diagnostic fragment ions. The carboxylate derivatization strategy in combination with ETD for proteomics applications by the proteolytic digestion, the derivatization, and LC-MS purification was demonstrated with Cytochrome C.

A series of studies from our lab have investigated the threading polyintercalator approach to sequence specific DNA binding using a 1,4,5,8-naphthalene tetracarboxylic diimide (NDI) intercalating unit connected by flexible peptide linkers. Herein is a report of the sequence specificity, as well as a detailed kinetic analysis, of a threading NDI tetraintercalator. DNase I footprinting using two ~500 base pair DNA fragments containing one designed binding site for the tetraintercalator confirmed highly sequence specific binding. Kinetic analyses include 1H NMR, gel mobility-shift assays, and stopped-flow UV measurements to reveal a polyintercalation binding mode that demonstrates significant similarities between association rate profiles and rate constants for the tetraintercalator binding to its preferred versus a random oligonucleotide sequence. Sequence specificity was found to derive almost entirely from large differences in dissociation rates from the preferred versus random oligonucleotide sequences. Interestingly, the dissociation rate constant of the tetraintercalator complex dissociating from its preferred binding site was extremely slow, corresponding to a 16 day half-life at a benchmark 100 mM [Na+]. This dissociation result for the tetraintercalator is one of the longest bound half-lives yet measured, and to the best of our knowledge, the longest for a DNA binding small molecule. Such a long-lived complex raises the possibility of using threading polyintercalators to disrupt biological processes for extended periods. Current focus is given to deciphering a mechanism for the molecular recognition of the tetraintercalator preferred binding site within a long sequence of DNA. Initial DNase I footprinting results on an approximate 500mer DNA sequence containing three sequential preferred binding sites reveal that the tetraintercalator likely locates its designed binding site by a macro- or microscopic dissociation/re-association type of mechanism. Cooperativity is a possible ally to binding, leaving future studies to distinguish the mechanism for molecular recognition in a manner that is capable of circumventing cooperative binding. Taken together, the threading polyintercalation binding mode presents an interesting topology to sequence specific DNA binding. Extraordinarily long dissociation rates from preferred binding sites offers many future possibilities to disrupt biological processes in vivo. / text

The research described in this dissertation is divided into two sections. The first section focuses on mass spectrometry-based bottom-up proteomics application to identify fungal protein biomarkers of invasive aspergillosis infection. The second part focuses on instrument development to improve current ionization and dissociation technologies for characterizing topology and substructure of protein complexes. Part I of this dissertation describes the identification and validation of protein biomarkers for Invasive Aspergillosis (IA), a fatal pulmonary infection. Aspergillus fumigatus, the organism responsible for this disease, is an opportunistic fungus. Immunocompromised individuals can suffer from IA due to impaired immune response. The current diagnostic tools are time-consuming and have variable sensitivity and specificity. Hence, treatments for IA are often administered too late. The goal of this research is to use mass spectrometry to identify and validate novel fungal protein biomarkers for IA. To tackle this challenge, several systems were studied. Commercial Aspergillus antigen was used for method development, and to serve as standards for spiking and comparison. Mouse models of different disease manifestations were used in the initial study to compare proteomic differences in carefully controlled disease states. Although it was not successful in providing candidate biomarkers, the mouse samples provided host response protein data. Human patient samples yielded the most promising results. Several Aspergillus proteins have been identified and validated from patient bronchoalveolar lavage fluid, and could have the potential to be later used on a diagnostic platform. Part II describes two instrument development projects: incorporation of a surface-induced dissociation device into a commercial ion mobility time-of-flight mass spectrometer, and the development of a paper spray ionization source. Protein complexes are often studied using collision-induced dissociation (CID), which does not provide enough substructure information. Surface-induced dissociation (SID) allows access to higher energy fragmentation pathways, which generates more useful substructure information. Its potential is demonstrated with three systems here-- one metal cluster and two protein complexes. All systems show that SID can provide more useful structural information than CID under similar conditions. The development of a paper spray (PS) source for protein complex ionization provides another way to study protein complexes. Chapter 9 shows that this ionization method can also be applied to protein complexes. Under the same conditions as its nanospray counterpart, similar mass spectra can be obtained using PS. This exciting result is the first demonstrations that PS can be used for protein complexes while maintaining each protein complex's native structure and conformation.

This dissertation presents dissociation mechanism and dissociation kinetics studies of gas-phase ions using mass spectrometry (MS). Dissociation of a gas-phase ion is related to its fundamental properties such as composition and structure. However, the detailed processes, internal energy deposition during ion activation as well as the mechanism of dissociation, are not fully known. In the present work, ion structural studies from which mechanisms can be inferred were performed using infrared multiphoton dissociation (IRMPD) spectroscopy, soft-landing, IR spectroscopy, and quantum chemical calculations. Kinetics studies involved instrument modification to add surface-induced dissociation (SID) capability and peak shape analysis. Structural studies were performed to determine dissociation mechanisms. The b₂⁺ ion from AGG is an oxazolone structure as indicated by the IRMPD spectrum and quantum chemical calculations. Protonated 4-ethoxymethylene-2-phenyl-2-oxazolin-5- one is also an oxazolone-type structure, while protonated cyclo-AG is a diketopiperazine structure. Soft-landing experiments were carried out to corroborate IRMPD results. Soft-landed protonated cyclo-AG and protonated 4-ethoxymethylene-2-phenyl-2- oxazolin-5-one underwent neutralization and retained their structures. The soft-landed b₂⁺ ion of AGG showed evidence of ring opening and conversion into a linear structure. The modified matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometers with SID capability were used to study fast dissociation kinetics (sub-microsecond dissociation). Silicon nanoparticle assisted laser desorption/ionization (SPALDI) allows the study of small molecule dissociation kinetics for ions without the matrix interference observed in MALDI. Well characterized systems, such as, N(CH₃)₄⁺, N(CD₃)₄⁺, and substituted benzylpyridinium ions were used to confirm reliability of the peak shape analysis. Obtained dissociation rates, of submicrosecond order, are consistent with the known dissociation theories. Dissociation of fullerenes, C₆₀ and C₇₀, was also investigated with the SID method using a fluorocarbon self-assembled monolayer (FSAM) surface. Fullerene ions produced C(2n)⁺ fragments ion in the kinetic energy range of 150-300 eV. At higher than 400 eV, mass spectra showed additional small fragment ions composed of odd numbers of C units. Energy resolved MS/MS curves support parallel dissociation at high SID energies while peak shape analysis explains sequential dissociation at about 150 eV range. Instrument modification of a MALDI-TOF mass spectrometer with SID capability allowed successful studies of fast unimolecular dissociation kinetics of small ions and fullerenes.

Understanding the intermolecular forces and dynamics of insulin self-assembly is crucial for devising formulations for the treatment of insulin-dependent diabetes. Insulin must dissociate from its hexameric storage form, through an intermediate dimer form, to the bioactive monomer before receptor binding. Specifically, the dimer dissociation is a pivotal step to control insulin dynamics and self-assembly. Steered molecular dynamics simulations were performed on native insulin to provide molecular insight into the insulin dissociation force spectroscopy experiment. Our simulation results of force-induced dimer dissociation revealed that the dimer dissociation occurs near the limit of extensibility of the B-chain with significant conformational changes to the monomer(s). These long-range interactions, consistent with our experiments, are due to stronger inter-monomer interactions across the anti-parallel β-sheet interface than any other intra-monomer interaction. Novel atomistic data played an important role in detailed structural characterization of multiple unfolding and dissociation pathways that depend on the relative strength of the inter-monomer interactions and the intra-monomer interactions. Comparative simulations of two rapid-acting insulin analogues (LysB28ProB29, AspB28) to native insulin were performed to investigate the effect of sequence on the dimer dissociation. The hypothesis is that site-specific alterations to the dimer-forming surface of two rapid-acting analogues will result in a weakening of the inter-monomer interactions, which would be reflected during force-induced dimer dissociation. The results revealed that these analogues dissociates with lower probability of long-range interactions and a corresponding reduction in B-chain extension. B-chain extensibility is thus a characteristic marker of inter-monomer interactions and multiple unfolding pathways. These data agree with the design strategies of sequence modifications to the weakened inter-monomer interface applied to the synthesis of rapid-acting insulin analogues. In contrast, the ligand-induced alteration to the strengthened inter-monomer interactions through a specific GluB13s-zinc bridge contributed to the unique unfolding force curves, so it can be applicable as design strategy to the development of a novel long-acting analogue. Overall, our force spectroscopy studies on insulin native and analogues have successfully provided atomistic insights into the dimer dissociation characteristics and control strategies of self-assembly. In addition, this study would provide a framework for the structure-dynamics-function relationships of insulin-insulin receptor binding.