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Reducing interferences in glow discharge spectroscopies using transient operation and collision-induced dissociationJackson, Glen P. January 1900 (has links)
Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xvii, 252 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.
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Mass spectrometric analysis of the kinetics of in vivo rhodopsin phosphorylation during light adaptation and recovery /Lee, Kimberly Alice. January 2003 (has links)
Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 92-98).
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Fundamental studies of electrothermal vaporization as a sample introduction source for inductively coupled plasma mass spectrometry /Venable, John Delmas, January 2001 (has links)
Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also in a digital version from Dissertation Abstracts.
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Interactions of anticancer therapeutics with DNA investigated via mass spectrometrySilvestri, Catherine Jane, 1985- 29 October 2012 (has links)
Many chemotherapeutic drugs interact with DNA to induce cytotoxicity. Mass spectrometry has become an essential technique in the investigation and identification of anticancer DNA adducts. Traditionally, identification of therapeutic DNA adducts was conducted by P1 enzymatic digestion followed by separation via gel electrophoresis or high performance liquid chromatography (HPLC). Structural information about binding was identified via NMR and x-ray crystallography. These methods are arduous and require significant sample consumption. Mass spectrometry is a high-through put methodology that requires a minimal amount of sample consumption to produce site specific binding information.
Anticancer agents may bind directly to DNA via formation of a covalent bond creating a monoadduct, a single covalent bond at one site of double stranded DNA, or a crosslink, two covalent bonds on each strand of duplex DNA. Cytotoxicity of covalent anticancer agents is achieved by effectively blocking replication of DNA, thus preventing proliferation of cancerous cells. Much effort has been directed in the search for new chemotherapies to increase binding specificity for cancerous cells. In designing new drugs it is essential to understand DNA interactive properties--such as differences in the cellular conditions, the number of nucleobases within the binding site, and the tendency to form a monoadduct or a crosslink. These factors can then be exploited to design more selective anti-cancer drugs.
Several covalent bond-forming anti-cancer DNA adducts have been investigated using mass spectrometry. These include mitomycin C, nitrogen mustards, cisplatin, psoralen derivatives, a bioreductive prodrug (RH1), and an enediyene. Mitomycin C is an anticancer antibiotic that forms a DNA crosslink at the 2-amino group of guanine. The DNA/mitomycin C adduct was evaluated by tandem mass spectrometry and the results demonstrated that the mitomycin C adduct formed isomeric tetramer nucleotides upon activation for dissociation.. Nitrogen mustards can form DNA crosslinks and monoadducts. The extent of DNA alkylation with a sulfur acridine mustard derivative was evaluated by tandem mass spectrometry using infrared multiphoton dissociation. . Cisplatin is an anticancer therapeutic that crosslinks DNA at N7 guanine residues. The fragmentation pattern of cisplatin/DNA adducts investigated by tandem mass spectrometry confirmed the formation of a crosslink in the platinated diagnostic fragment ions detected. Psoralens, used for centuries to treat psoriasis, form crosslinks preferentially at thymine nucleobases by the sequential absorption of two photons. The results of tandem mass spectrometry were used to identify the sequence selectivity of psoralen derivatives. Recently, a study of a bioreductive prodrug, 2,5-diaziridinyl-3-[hydroxymethyl]-6-methyl-1,4-benzoquinone (RH1), was shown to form crosslinks with DNA at N7 guanine residues and the fragment ions produced via tandem mass spectrometry confirmed the site of the crosslink. Lastly, enediynes are of therapeutic interest because they exhibit a high cytotoxicity when the drug moiety forms a DNA crosslink through a biradical intermediate across opposing cytosine nucleobases. The tandem mass spectrometry results indicated that the enediyne moiety binds in a somewhat nonselective manner, as it associated with thymine as well as cytosine, and the formation of a covalent crosslink was confirmed by the retention of the enediyne by diagnostic fragment ions. .
Other anticancer agents associate with DNA through noncovalent interactions like minor groove binding or intercalation. In both cases, the electrostatic interactions between the chemotherapeutic agent and the DNA double helix interfere with DNA transcription leading to incomplete proteins synthesis and ultimately cell death. Noncovalent anticancer moieties historically suffer from a lack of specificity, as most drug moieties have only two to four base pair binding sites. Thus, current research focuses on increasing the specificity of these small molecules.
A novel tetraintercalator, 1,4,5,8-tetracarboxylic naphthalene diimide units connect by peptides (TET), has four intercalation units and a 14 base pair binding site allowing for dramatically greater sequence selectivity. The novel tetraintercalator shows the highest specificity amongst known intercalating moieties. An investigation into the sequence selectivity and binding site affinity compared to well characterized small molecule intercalators, actinomycin D and echinomycin, was assessed by mass spectrometry. The results show that TET preferentially binds to sequences that contain the unmodified binding site and also shows a slight preference to adenine and thymine rich sequences, indicating the peptide linkers play an important role in DNA interactions. Tandem mass spectrometry results demonstrated that TET binds with high affinity to its binding site compared to small molecule intercalators. Upon collision induced dissociation (CID) the predominant species in the mass spectrum was the DNA/TET – G ion peak. Intercalating adducts generally dissociate by strand scission with either strand retaining the drug moiety or by ejection of the drug, as seen with both actinomycin D and echinomycin in this study. Therefore, TET shows promise as a new development toward an anticancer therapeutic with high sequence selectivity and binding affinity with DNA.
This workfocuses on reviewing the advancements of covalent bond forming DNA interactive anticancer therapeutics that have been studied by mass spectrometry, and presents a study of the interactions of a novel intercalation drug with DNA explored by mass spectrometry. / text
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Multidimensional liquid chromatography for the analyses of hydrophilicand hydrophobic components in mass spectrometry-based proteomicsLam, Pui-yu., 林沛瑜. January 2011 (has links)
published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
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Development of fully automatable multidimensional liquid chromatography (MDLC) with online tandem mass spectrometry for shotgunproteomicsKong, Pak-wing., 江柏榮. January 2011 (has links)
Proteomics is the systematic study of the proteome: the total protein expression of a cell or tissue under specified conditions. The multiplicity and complexity of proteins in cells requires sensitive, selective, and comprehensive methodologies for their distinction and characterization. Multidimensional liquid chromatography (MDLC) coupled with biological tandem mass spectrometry (MS/MS) is uniquely suited to fulfill those requirements and has become an indispensable tool in MS-based proteomics. Our laboratory has developed an online high-/low-pH reversed-phase/reversed-phase (RP–RP) LC system exhibiting fully automatable and reproducible performance. It is a promising alternative to the strong cation exchange/reversed-phase (SCX–RP) system commonly used in high-throughput comprehensive proteomics analyses. The first part of this Thesis (Chapter 2) describes the development of a variant of the high-/low-pH RP–RP platform—RP–SCX–RP—that integrates an additional SCX trap column between the two RP columns to enhance sample recovery. This new system allows the detection of larger numbers of hydrophilic peptides. Indeed, in the analyses of a lysate of Arabidopsis chloroplast proteins, it identified approximately 25% more non-redundant proteins than those identified using the previous version of the RP–RP system. The modified platform has been extended for the online removal of sodium dodecyl sulfate and other excess interference chemicals used in Isobaric Tags for Relative and Absolute Quantification (iTRAQ) reactions, thereby avoiding the need for time-consuming offline SCX clean-up prior to RP–RP separation in the quantitative proteomics analyses of crude biological samples at low-microgram levels. A novel online three-dimensional liquid chromatography (3DLC) system was derived from the RP–SCX–RP design, exhibiting remarkably enhanced orthogonality, resolution, and peak capacity. Peptides were separated in the first-dimension high-pH RP column based on their hydrophobicity, followed by sub-fractionation in the second-dimension SCX column, primarily based on charge; the third dimension was a typical low-pH RP separation, prior to MS analysis. The overall performance of the system was evaluated through analysis of a cell lysate of mouse embryonic fibroblasts. Relative to the two-dimensional high-/low-pH RP–RP system, the new 3D system yielded significant increases in the number of unique peptides and proteins identified, making it a good alternative to SCX–RP and high-/low-pH RP–RP as an efficient automated MDLC platform for high-throughput shotgun proteomics. An optimized and miniaturized variant of the three-dimensional LC platform was also developed. Its simplified setup and operation, by decreasing the number of six-port switching valves (from three to two) and the number of SCX fractionation steps, minimized both the potential sample loss and the total analysis time (by ca. 30%). Thus, a variety of novel, automatable, and robust RP–SCX–RP-based MDLC platforms have been developed for high-throughput qualitative and quantitative analysis. The performance of these systems complements conventional MDLC systems, with enhanced quality, quantity, reproducibility, and throughput of protein identification and quantification. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
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Studies of single-particle inductively coupled plasma mass spectrometryLee, Wan-waan, 李雲鬟 January 2014 (has links)
abstract / Chemistry / Doctoral / Doctor of Philosophy
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Conducting multi-elemental analyses with an inductively coupled plasma mass spectrometer using electrothermal vaporization sampleBalsanek, William John 28 August 2008 (has links)
Not available / text
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Enhanced protein characterization through selective derivatization and electrospray ionization tandem mass spectrometryVasicek, Lisa Anne 07 November 2011 (has links)
There continue to be great strides in the field of proteomics but as samples become more complex, the ability to increase sequence coverage and confidence in the identification becomes more important. Several methods of derivatization have been developed that can be used in combination with tandem mass spectrometry to identify and characterize proteins. Three types of activation, including infrared multiphoton dissociation, ultraviolet photodissociation, and electron transfer dissociation, are enhanced in this dissertation and compared to the conventional method of collisional induced dissociation (CID) to demonstrate the improved characterization of proteins.
A free amine reactive phosphate group was synthesized and used to modify the N-terminus of digested peptides. This phosphate group absorbs at the IR wavelength of 10.6 µm as well as the Vacuum-ultraviolet (VUV) due to an aromatic group allowing modified peptides to be dissociated by infrared multi-photon dissociation (IRMPD) or ultraviolet photodissociation (UVPD) whereas peptides without this chromophore are less responsive to IR or UV irradiation. The PD spectra for these modified peptides yield simplified MS/MS spectra due to the neutralization of all N-terminal product ions from the incorporation the negatively charged phosphate moiety. This is especially advantageous for UVPD due to the great number of product ions produced due to the higher energy deposition of the UV photons. The MS/MS spectra also produce higher sequence coverage in comparison to CID of the modified or unmodified peptides due to more informative fragmentation pathways generated upon PD from secondary dissociation and an increased ion trapping mass range.
IRMPD is also implemented for the first time on an orbitrap mass spectrometer to achieve high resolution analysis of IR chromophore-derivatized samples as well as top-down analysis of unmodified proteins. High resolution/high mass accuracy analysis is extremely beneficial for characterization of complex samples due to the likelihood of false positives at lower resolutions/accuracies.
For electron transfer dissociation, precursor ions in higher charge states undergo more exothermic electron transfer and thus minimize non-dissociative charge reduction. In this dissertation, cysteine side chains are alkylated with a fixed charge to deliberately increase the charge states of peptides and improve electron transfer dissociation. ETD can also be used to study protein structure by derivatizing the intact structure with a hydrazone reagent. A hydrazone bond will be preferentially cleaved during ETD facilitating the recognition of any modified residues through a distinguishing ETD fragmentation spectrum. / text
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Investigating chaperone dynamics : mass spectrometry as a tool for solving protein complex architecture and dynamicsStengel, Florian January 2010 (has links)
No description available.
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