Thesis (Ph. D.)--University of California, Riverside, 2009. / Includes abstract. Title from first page of PDF file (viewed March 8, 2010). Includes bibliographical references. Issued in print and online. Available via ProQuest Digital Dissertations.

Gas electron diffraction (GED) is a technique that has been developed to study the molecular structure of species in the gas phase. This thesis focuses on the reconstruction of the Canterbury GED apparatus (moved from Edinburgh, UK) and the requirements for modifying the apparatus to incorporate a mass spectrometer (MS) so diffraction and MS data can be obtained within a single experiment. The combined GED-MS system has been identified in previous work in the Masters group as a necessary development for studying the structure of short-lived species generated in situ. This is particularly true for the study of ketene, which as shown in this thesis, can be generated from several precursors as part of a multiple product pyrolysis system. While GED data for ketene generated from acetic anhydride has been refined, the species formed from the pyrolysis of Meldrum’s acid were determined to be too difficult to deconvolute without additional experimental data from MS. A computational study of possible ketene derivatives that could be studied with a GED-MS apparatus is also presented. Lastly, this thesis details a structural study of the gas-phase structures of tris(chloromethyl)amine and a family of substituted disilane systems which have been determined in the gas phase for the first time. A comprehensive GED, Raman spectroscopy and ab initio study have been undertaken for tris(chloromethyl)amine [N(CH2Cl)3] which is shown to have a different structure in the solid and gas phase. Further work in the form of a molecular dynamics investigation has been identified as necessary to describe the low amplitude motion of one of the CH2Cl groups in the gas phase to allow for the GED refinement to be completed. The work on the substituted disilane systems X3SiSiXMe2 (X = F, Cl, Br, I) and X3SiSiMe3 (X = H, F, Cl, Br) demonstrates the effect of increased halogen substitution on the electronic effects of the disilanes, and the effect that the methyl groups have as larger halogens increase the steric bulk of the system.

Matrix-assisted laser desorption/ionization (MALDI) is a technique used in mass spectrometry for the ionization of biomolecules. A matrix solution is mixed with the analyte molecules to be investigated, and then spotted onto a specialized MALDI plate. The solvents evaporate leaving only the re-crystallized matrix with analyte dispersed throughout the crystals. Sample ionization is accomplished with a laser in the MALDI instrument. The spot diameter of the target is usually several orders of magnitude larger than the diameter of the laser, making it necessary to perform multiple laser investigations to accurately evaluate the analyte in the target spot. Experiments were performed to utilize patterned areas of carbon nanotubes to provide sites for preferential crystallization of the liquid matrix/analyte solution, which led to lateral concentration for non-aqueous based matrices and produced a final dried matrix/analyte spot that was approximately the diameter of the laser spot at the point of investigation. This work shows the results of using aligned carbon nanotubes as the substrate for the matrix/analyte deposition and demonstrates an increase in signal to noise ratio and an improved detection capability of low analyte concentrations compared to the standard MALDI preparation technique.

Proteomics is a fast growing field and there has been a tremendous increase of knowledge the last two decades. Mass spectrometry is the most used method for analysis of complex protein samples. It can be used both in large scale discovery studies as well as in targeted quantitative studies. In parallel with the fast improvements of mass spectrometry-based proteomics there has been a fast growth of affinity-based methods. A common challenge is the large dynamic range of protein concentrations in biological samples. No method can today cover the whole dynamic range. If affinity and mass spectrometry-based proteomics could be used in better combination, this would be partly solved. The challenge for affinity-based proteomics is the poor specificity that has been seen for many of the commercially available antibodies. In mass spectrometry, the challenges are sensitivity and sample throughput. In this thesis, large scale approaches for validation of antibodies and other binders are presented. Protein microarrays were used in four validation studies and one was based on mass spectrometry. It is shown that protein microarrays can be valuable tools to check the specificity of antibodies produced in a large scale production. Mass spectrometry was shown to give similar results as Western blot and Immunohistochemistry regarding specificity, but did also provide useful information about which other proteins that were bound to the antibody. Mass spectrometry has many applications and in this thesis two methods contributing with new knowledge in animal proteomics are presented. A combination of high affinity depletion, SDS PAGE and mass spectrometry revealed 983 proteins in dog cerebrospinal fluid, of which 801 were marked as uncharacterized in UniProt. A targeted quantitative study of cat serum based on parallel reaction monitoring showed that mass spectrometry can be an applicable method instead of ELISA in animal proteomic studies. Mass spectrometry is a generic method and has the advantage of shorter and less expensive development costs for specific assays that are not hampered by cross-reactivity. Mass spectrometry supported by affinity based applications will be an attractive tool for further improvements in the proteomic field.

Based on reactions with two flavanones, three flavonols, and five flavones the regioselectivities of twelve human UDP-glucuronosyltransferase (UGT) isozymes were elucidated. The various flavonoid glucuronides were differentiated based on LC-MS/MS fragmentation patterns of [Co(II)(flavonoid – H)(4,7-diphenyl-1,10-phenanthroline)2]+ complexes generated upon post-column complexation. Glucuronide distributions were evaluated to allow a systematic assessment of the regioselectivity of each isozyme. The various UGT enzymes, including eight UGT1A and four UGT2B, displayed a remarkable range of selectivities, both in terms of the positions of glucuronidation and relative reactivity with flavanones, flavonols and flavones. The UGT1A enzyme selectivities are affected by the presence of a hydroxyl group at the 3, 6, 4’, or 3’ positions as well as by the presence of a methoxy at the 3’ position. The UGT2B enzymes show poor to no reactivity with the flavonols or flavones. This result implies that the greater planarity of the flavonols and flavones compared to structure of flavanones inhibits interaction with the UGT2 enzymes. For baicalein and scutellarein, three of the UGT1A isozymes (1A8, 1A9, and 1A10) resulted in the formation of 6-O glucuronides, enabling the fragmentation rules for the metal complexation/MS/MS strategy to be expanded. / 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.

Many anticancer and antibacterial therapies are based on the interaction of small molecule drugs with DNA. Increasing interest in the development of DNA-interactive agents has fostered the need for sensitive and versatile analytical techniques that are capable of characterizing the DNA/ligand interactions and are compatible with librarybased screening methods. Electrospray ionization mass spectrometry (ESI-MS) has emerged as a useful technique for the analysis of non-covalent complexes formed between DNA and small molecules due to its low sample consumption and fast analysis time. The work presented in this dissertation is aimed at exploring, optimizing, and validating ESI-MS methods for characterizing DNA-ligand interactions. ESI-MS is first used to assess the binding of threading bis-intercalators to duplexes containing different sequences to determine high affinity binding sites of the ligands. Preliminary DNAse footprinting experiments identified possible specific binding sites of the ligands and ESI-MS experiments revealed that the ligands bound to DNA duplexes containing the respective specific binding sequences. The metal-mediated binding of benzoxazole ligands with different side chains to duplex DNA is also examined. Cu²⁺ and Ni⁺ were found to promote the most dramatic increase in ligand binding, and ligands exhibiting the most dramatic metal-mediated or metal-enhanced binding were also determined to be the most cytotoxic. The quadruplex DNA binding selectivity of perylene diimides is evaluated by screening the binding of the ligands to quadruplex, duplex and single strand DNA by ESI-MS. Three ligands, one containing basic side chains, one containing anionic sidechains, and one benzannulated compound were determined to be the most-quadruplex selective. The ESI-MS results correlated well with spectroscopic experiments. The relative gas-phase stabilities of different quadruplex DNA structures were investigated using molecular dynamics simulations and ESI-MS. The stabilities from the E[subscript 1/2] values generally paralleled the RMSD and relative free energies of the quadruplexes based on MD energy analysis. Finally an ESI-MS technique employing the KMnO₄ reaction with DNA to determine conformational changes to the duplex structure upon ligand binding is detailed. Thymines in most intercalator/duplex complexes are more susceptible to oxidation by KMnO₄ than those in duplex DNA. CAD and IRMPD experiments are used to identify the site of oxidation. / text

Several innovative tandem mass spectrometric strategies have been developed for the structural determination and isomer differentiation of glycosides and oligosaccharides. Specifically, collisionally activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) are used in conjunction with derivatization methods designed to exploit variations in binding energies or attach chromophores. These include metal complexation incorporating modified neutral auxiliary ligands and covalent derivatization involving site-specific reactions. The elucidation of flavonoid isomers is accomplished by electrospray ionization tandem mass spectrometry (ESI-MS/MS) via formation and CAD of metal/flavonoid complexes containing an auxiliary ligand. Addition of a metal salt and a suitable neutral auxiliary ligand to flavonoids in solution results in the formation of [M(II) (flavonoid-H) ligand]⁺ complexes by ESI which, upon collisional activated dissociation, often result in more distinctive fragmentation patterns than observed for conventional protonated or deprotonated flavonoids. We compare and explore the use of alternative pyridyl ligands, with electron-releasing substituents including 4,7-diphenyl-1,10-phenanthroline. Using this technique, three groups of flavonoid glycoside isomers are differentiated, including glycosides of apigenin, quercetin and luteolin. A tunable ESI-MS/MS strategy for differentiation of flavone and flavanone diglycoside isomers based on metal complexation with auxiliary ligands that have electron-withdrawing substituents is reported. A series of auxiliary ligands with electronwithdrawing substituents was synthesized in order to tailor the relative metal binding affinities of the ligands and thus directly influence the stabilities, and consequently the dissociation pathways, of the complexes. Upon collisionally activated dissociation, the complexes yield fragmentation patterns in which the abundances of key diagnostic ions are enhanced, thus facilitating isomer differentiation. A strategy for increasing the efficiency of IRMPD in a quadrupole ion trap (QIT) based on another metal complexation strategy is described. Two IR-active ligands (IRALs) that have an IR-active phosphonate functional groups were synthesized. The IR-active groups were therefore incorporated into the analyte complexes via metal complexation. We demonstrate this new IRMPD approach for the structural characterization of flavonoids. The fragment ions obtained by IRMPD are similar to those obtained by CAD and allow facile isomer differentiation of flavonoids. Fourier transform infrared absorption attenuated total reflectance (FTIR-ATR) and energyvariable CAD experiments indicate that the high IRMPD efficiencies stem from the very large IR absorptivities of the IR-active ligands. A simplified method for determining the sequence and branching of oligosaccharides using IRMPD in a QIT is described. An IR-active boronic acid (IRABA) reagent was synthesized and subsequently used to derivatize the oligosaccharides prior to IRMPD analysis. The IRABA ligand is designed to both enhance the efficiency of the derivatization reaction and to facilitate the photon absorption process. The resulting IRMPD spectra display oligosaccharide fragments that are formed from primarily one type of diagnostic cleavage, thus making sequencing straightforward. The presence of sequential fragment ions, a phenomenon of IRMPD, permit the comprehensive sequencing of the oligosaccharides studied in a single stage of activation. The approach is demonstrated for two series of oligosaccharides, the lacto-Nfucopentaoses (LNFPs) and the lacto-N-difucohexaoses (LNDFHs).

Plants, green algae and cyanobacteria perform photosynthetic conversion of sunlight into chemical energy in the permanently changing natural environment. For successful survival and growth photosynthetic organisms have developed complex sensing and signaling acclimation mechanisms. The environmentally dependent protein phosphorylation in photosynthetic membranes is implied in the adaptive responses; however, the molecular mechanisms of this regulation are still largely unknown. We used a mass spectrometry-based approach to achieve a comprehensive mapping of the in vivo protein phosphorylation sites within photosynthetic membranes from the green alga Chlamydomonas reinhardtii subjected to distinct environmental conditions known to affect the photosynthetic machinery. The state transitions process regulating the energy distribution between two photosystems, involves the temporal functional coupling of phosphorylated light-harvesting complexes II (LHCII) to photosystem I (PSI). During state transitions several of the thylakoid proteins undergo redox-controlled phosphorylation-dephosphorylation cycles. This work provided evidences suggesting that redox-dependent phosphorylation-induced structural changes of the minor LHCII antenna protein CP29 determine the affinity of LHCII for either of the two photosystems. In state 1 the doubly phosphorylated CP29 acts as a linker between the photosystem II (PSII) core and the trimeric LHCII whereas in state 2 this quadruply phosphorylated CP29 would migrate to PSI on the PsaH side and provide the docking of LHCII trimers to the PSI complex. Moreover, this study revealed that exposure of Chlamydomonas cells to high light stress caused hyperphosphorylation of CP29 at seven distinct residues and suggested that high light-induced hyperphosphorylation of CP29 may uncouple this protein together with LHCII from both photosystems to minimize the damaging effects of excess light. Reversible phosphorylation of the PSII reaction center proteins was shown to be essential for the maintenance of active PSII under high light stress. Particularly dephosphorylation of the light-damaged D1 protein, a central functional subunit of the PSII reaction center, is required for its degradation and replacement. We found in the alga the reversible D1 protein phosphorylation, which until our work, has been considered as plant-specific. We also discovered specific induction of thylakoid protein phosphorylation during adaptation of alga to limiting environmental CO2. One of the phosphorylated proteins has five phosphorylation sites at both serine and treonine residues. The discovered specific low-CO2- and redox-dependent protein phosphorylation may be an early adaptive and signalling response of the green alga to limitation in inorganic carbon. This work provides the first comprehensive insight into the network of environmentally regulated protein phosphorylation in algal photosynthetic membranes.

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