O trabalho realizado teve como objetivo principal o projeto, a construção e os testes de um sistema de vácuo dedicado a técnica de Espectrometria de Massa do íon Secundário (SIMS), a ser utilizado na caracterização estrutural de heteroestruturas semicondutoras. O sistema (câmaras e conexões) foi totalmente desenvolvido nas dependências do IFSC-USP, sendo acoplado a este um conjunto de elementos comerciais, tais como: visores de Ultra-Alto Vácuo (UHV), medidores, canhão de íons, válvulas, bombas (mecânica, turbo e iônica) e espectrômetro de massa. Testes realizados mostraram que o sistema de vácuo principal (o sistema todo é composto por duas câmaras de vácuo: uma principal e uma de introdução) pode atingir pressões da ordem de 10-10 Torr, necessária para a implementação de técnicas de análise de superfície. Além disto, o sistema mostrou-se versátil para a troca de amostras (uso continuo) e de fácil operação. Além de instrumentação para UHV, os resultados obtidos são promissores no sentido de proporcionar uma economia nos custos para futuras aquisições de sistemas para analise de superfícies / The principal purpose of this work was the project, construction and test of a vacuum system dedicated to Secondary Ion Mass Spectroscopy (SIMS), for the structural characterization of semiconductor heterostructures. All the system components were developed at the IFSC-USP, to which were connected commercial vacuum elements, such: Ultra-high Vacuum (UHV) visors, sensors, ion gun, vacuum pumps (mechanical, turbo and ionic) and mass spectrometer. The results showed that the main chamber (the system is composed by two chambers: main chamber and the load-look) is able to reach pressures of 10-10 Torr, important for surface analyses. Also the system showed versatility to change the samples and easy operation. The results for UHV instrumentation developed are promising, and these results can be used to decrease the cost of surface analysis systems which can be implemented in the future

No description available.

Protein-nucleic acid interactions play essential roles in all living cells in various cellular functions. The study of these interactions can reveal important structural and functional information. UV cross-linking of nucleic acids to proteins in combination with mass spectrometry is a powerful technique to identify proteins, peptides and the amino acids involved in intermolecular interactions within nucleic acid-protein complexes. However, the mass spectrometric identification of cross-linked nucleic acid-protein heteroconjugates in complex mixtures and MS/MS characterisation of the specific sites of cross-linking is a challenging task. In this investigation, novel tools and methods have been developed for the investigation of DNA-protein interactions using UV cross-linking and mass spectrometry. These tools were developed towards their application for the characterisation of the complex between the eukaryotic DNA repair protein Tyrosyl-DNA phosphodiesterase 1 (Tdp1) and its DNA substrates. DNA-Tdp1 UV cross-linking was optimised using purified recombinant human Tdp1 and radioactively labelled DNA oligonucleotides containing UV photoactivatable 4- thio-thymidine or 5-iodouracil. Tdp1-DNA heteroconjugates were detected by SDS PAGE and Phosphorimaging. In order to analyse the DNA-Tdp1 heteroconjugates by mass spectrometry, they must first be enriched and hydrolysed by a protease and a nuclease. Here, a novel sample preparation protocol was developed for the enrichment of Tdp1 oligonucleotide-peptide heteroconjugates. Detection and analysis of oligonucleotide-peptide heteroconjugates using mass spectrometry is a challenging task. As a tool to optimise the various parameters involved, a synthetic DNA oligonucleotide-peptide heteroconjugate was constructed using click chemistry. RP-HPLC/ESI-FT-ICR-MS on a Bruker 12T SolariX in conjunction with CID fragmentation was used to unambiguously identify the site of the cross-link. Lastly, a novel 18O labeling approach was introduced to facilitate the identification of DNA-protein cross-links. This approach was shown to be suitable for the labeling of heteroconjugate species by testing it with the click heteroconjugate.

Glycosaminoglycans (GAGs) are linear polysaccharides found on most animal cell surfaces and in extracellular matrices. Their key biological roles include cell signalling, cell-to-cell recognition, bacterial and viral adhesion, and antibody production. They are composed of disaccharide repeating units, containing uronic acid and amino sugar residues, and may be highly heterogeneously sulfated. Protein-GAG complexes are thought to play an important role in a number of aspects of cancer development, but are an under-studied area due to a lack of enabling tools to facilitate their analysis as discussed in Chapter 1. To obtain homogenous GAG structures, a range of conditions for the separation of GAG oligosaccharides by Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) were developed using commercial chondroitin sulfate standards; these are discussed in Chapter 2. Mixed-modal separation mechanisms were explored across a different buffer compositions and elution programs to optimise the conditions to suit individual classes of native and modified glycosaminoglycans. These methods were then applied to the separation of chondroitin sulfate mixtures and heparin oligosaccharides. ZIC-HILIC may also be coupled to Mass Spectrometry to produce an online analytical method for GAG mixtures. A range of optimised conditions for the analysis of low molecular weight glycosaminoglycan oligosaccharides by Electrospray Mass Spectrometry were developed using commercial chondroitin sulfate disaccharide standards; as discussed in Chapter 3. These conditions were then employed in the tandem mass spectrometry (MS/MS) of chondroitin sulfate to identify diagnostic fragmentation patterns and this was applied in the characterisation of chondroitin sulfate disaccharides derived from enzymatic cleavage. The position of ring substituents can also be identified using this methodology, which is desirable in the analysis of chemically-labelled GAG structures. To allow for high resolution EPR and NMR studies of protein-GAG interactions, synthetic procedures for the incorporation of paramagnetic centres into GAG oligosaccharides and proteins were developed and these are discussed in Chapter 4. A propargyl-modified lysine was synthesised for recombinant expression into myoglobin. Copper-Catalysed Azide-Alkyne Cycloaddition (CuAAC) was employed in the spin labelling of myoglobin, however traditional experimental conditions were found to reduce the TEMPO-based spin labels used. Alternative conditions were developed using glutathione to stabilise the copper (II) catalyst without reducing the spin label. Spin labelling of the modified lysine was monitored by EPR. Modification of the non-reducing end was explored for the spin labelling of GAG oligosaccharides, to avoid the ring opening that typically occurs in reducing end labelling, and make use of the unsaturated uronic acid that is derived when obtaining GAGs through enzymatic depolymerisation. A hydrazide labelling of uronic acids was initially adopted, however due to concerns regarding selectivity and availability of hydrazide spin labels, an alternative Thiol-Ene Click (TEC) method was developed. TEC labelling of monosaccharides and unnatural amino acids was found to proceed efficiently under aqueous conditions and was monitored by NMR and HPLC. Preliminary studies in the TEC labelling of chondroitin sulfate disaccharides proved promising, however further optimisation is required for this method to be utilised in the study of protein-GAG interactions by NMR.

The ability to characterize the interactions between ions and solvent molecules plays a critical role in understanding fundamental aspects of thermodynamics in solution chemistry. These interactions are often difficult if not impossible to observe in solution due to the number of solvent molecules far exceeding that of the ions. However, this challenge can be circumvented in the gas phase which enables the isolation and study of reactions between a single ion and single solvent molecule. Within the field of ion-molecule chemistry are two sub-categories of interactions known as ionic hydrogen bonds (IHBs) and ionic halogen bonds (IXBs). In these interactions, the incorporation of a charged species permits ion-dipole interactions which are innately stronger than those found in dipole-dipole interactions among neutral molecules. This dissertation describes and explains the interactions which take place between halogenated benzenes (F-, Cl-, Br-, and Iodobenzene) and neutral polar molecules (water, acetonitrile, acetone, and methanol). Additional studies on ionic hydrogen bonding involve the exploration of protonated benzonitrile monomer and dimer solvated by methanol. All systems were examined using the mass-selected ion mobility technique using the VCU mass selected ion mobility mass spectrometer. Thermochemical equilibrium measurements, in conjunction with density functional theory (DFT) calculations, were performed, enabling comparison between experimentally and theoretically determined binding energies. Additionally, the DFT calculations were able to validate hypothetical predictions for the lowest energy structures of each interaction. Furthermore, the averaged collision cross sections of the benzonitrile dimer radical cation, protonated benzonitrile dimer, and benzonitrile solvated hydronium ion were elucidated using the technique of ion mobility where experimentally determined cross sections were compared with theoretical collision cross section calculations on predetermined geometries that were optimized using DFT calculations.

No description available.

Bibliography: leaves 134-148.

The aim of this project was to investigate a range of medicinal herbs which have radical scavenging and antioxidant activities and then apply novel mass spectrometric techniques to investigate and analyse active components responsible for their pharmaceutical actions. A sensitive electron capture negative ionization of gas chromatography-mass spectrometry (ECNI-GC-MS) method was developed to assess hydroxyl radical production, as indicated by 3.4-dihydroxyphenylacetic acid (DOPAC) production, which allows excellent evaluation of hydroxyl radical scavenging and antioxidant activity of a number of medicinal Chinese herbs. Melatonin is an effective multiple radical scavenger and antioxidant and has been used in this study for the comparison of radical scavenging activity with medicinal herbs. To analyse active compounds from herbal extracts, mass spectrometric techniques were used to separate components that suppressed hydroxyl radical production from Dimocarpus longan Lour, determine known ginsenosides from ginseng extracts as well as to identify and quantify melatonin in ten herbal extarcts. The results obtained indicated that 1) the utilization of alumina in the ECNI-GC-MS method diminished interferences from ???noise??? products in a Fenton-type reaction, which allows obtaining pure final hydroxyl radical product and this method demonstrated optimal sensitivity and reliability; 2) Aqueous extracts of all herbs analysed showed different levels of hydroxyl radical scavenging activity. Dimocarpus longan Lour, Chrysanthemum morifolium Ramat, Lonicera hypoglauca Miq, Ginkgo biloba L, Rehmannia flutinosa and Libosch Cornus officinalis Sieb all exhibited stronger inhibitory effect on hydroxyl radical production than melatonin. 3) Aqueous extract of Dimocarpus longan Lour. showed the greatest inhibitory effect on hydroxyl radical production among the other herbs tested. The active fractions of this herb eluted just after the void volume using HPLC suggesting that the active compounds responsible for radical scavenging activity are polar and water soluble. They may belong to phenol group of chemicals. 4) Herbal extracts using non-polar solvents showed no effect on hydroxyl radical production suggesting active compounds in those herbs are water soluble. 5) Different species and origins of ginseng were compared for their radical scavenging activity. Chinese fresh ginseng (Oriental ginseng) showed higher activity than Korean ginseng tablet and American ginseng. Seven known active ginsenosides were identified using HPLC-MS-MS. 6) Melatonin was found at varying concentrations in ten herbs, which may contribute to the radical scavenging activity of herbs, on the other hand, it may provide the justification of clinical use and food resources, particularly for those herbs contain high level of melatonin.

The performance of a linear matrix assisted laser desorption/ionization mass spectrometer (MALDI-oa-TOFMS) was improved with more reproducible sample preparation methods, a higher rate digitiser for integrating signals and customisable computer control, data acquisition and analysis in the LabVIEW?programming environment. This resulted in a ~20% improvement in resolution (up to 4,400) and enabled measurement of desorption velocities of 1,000 - 1,800 ms-1 for analytes with m/z 615 ?1,350 Da, with matrix ion velocities being 4,000 ?4,800 ms?. Detector limitations and restrictions on source axis energy (and hence velocity) required for the analysis of ions prevented detection of other species with this instrument. A 20 kV reflecting geometry MALDI-oa-TOFMS was constructed to overcome these limitations and extend the mass range. This mass spectrometer was able to analyse ions desorbed with a wide range of energies (32 ?197 eV). The resolution was found to be 8,000 -10,000. Best mass accuracy was 15-80 ppm (internal standards ). External calibration gave larger mass errors, mostly due to timing jitter, but the mass axis was stable for &lt2 weeks. Mass accuracy was independent of the analyte and matrix used. Ions with m/z of ~10,000 - 20,000 Da were observable with the use of a pulsed lens in the target region. This lens increased signal approximately 20 times, but degraded resolution. The detection limit of the instrument (sample consumed) was estimated to vary from 10 ?90 fmol, by extrapolation, with more moles required at higher m/z. The microsphere plate (MSP) electron multiplier used in the reflecting instrument was found to have a temporal response of &gt1 ns FWHM, but with a low secondary electron conversion efficiency, making it unsuitable for high m/z species. Experiments were also performed with a novel rectangular mesh grid, which (in correct orientation) provided similar resolution to conventional square mesh grids, but with significantly improved transmission and hence sensitivity.

Thesis (Ph. D.)--Brigham Young University. Dept. of Chemistry and Biochemistry, 2006. / Includes bibliographical references.