Browsing by Subject "Analytical chemistry"
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Item Development of ultraviolet photodissociation based tandem mass spectrometry methods for the characterization of protein macromolecular structures and glycolipids(2014-05) O'Brien, John Patrick, 1986-; Brodbelt, Jennifer S.; Crooks, Richard M; Webb, Lauren J; Trent, Stephen M; Browning, Karen S; Roberts , Sean TPhoton-based tandem mass spectrometry provides a versatile ion activation strategy for the analysis of polypeptides, proteins, and lipids. 351-nm ultraviolet photodissociation mass spectrometry (UVPD-MS) is a facile and selective tandem dissociation technique used to elucidate chromophore-modified peptides within large mixtures. A bis-aryl chromogenic chemical probe was utilized to target solvent exposed primary amine residues within native protein states. Collision-induced dissociation (CID) was employed to indiscriminatly characterize the complete proteolytic digest while chromophore containing peptides were selectively dissociated with 351-nm UVPD; thus streamlining the identification of targeted peptides with structurally informative residues. Protein amine residue reactivities were then compared with predicted solvent exposures to elucidate protein tertiary structures, their mechanistic properties, and ligand-binding interactions. High-energy 193-nm UVPD is a fast, high-energy tandem mass spectrometry method and frequently generates fragment ions typically inaccessible to CID-based methods. Native mass spectrometry was coupled to top-down 193-nm UVPD for the gas phase characterization of non-covalent protein-ligand and protein-protein complexes. This method yielded a unique array of fragment ions for a comprehensive analysis of protein structures. UVPD of non-covalent complexes generated many polypeptide backbone fragments to characterize the primary sequence of proteins. Furthermore, top-down UVPD engendered cleavages with intact electrostatic interactions; this provided insight into the binding interfaces within protein-ligand complexes and the higher order structural architectures of oligomeric complexes. High-resolution 193-nm UVPD was paired with high performance liquid chromatography (LC) for the streamlined structural analysis of amphiphilic glycolipids within complex mixtures. For all glycolipids, UVPD provided the most comprehensive structural analysis tool by affording a diverse array of fragment ions to characterize both hydrophobic and hydrophilic moieties. UVPD based LC-MS separations of gangliosides shed light on the ceramide lipid bases, glycan moieties, and their isobaric structural variants. UVPD activation of lipid A and lipooligosaccharides (LOS) compounds generated a mixture of C-C, C-O, and C-N fragment ions to illustrate the hydrophobic acyl structures, while cleavages within the glycosidic, and cross-ring cleavages allowed the determination of acylation patterns. Novel LC-MS separation strategies were developed to elucidate and structurally characterize complex mixtures of lipopolysaccharide containing compounds.Item Enzymatic inhibition-based biosensing on nitrogen-doped carbon nanotube electrodes(2015-05) Rust, Ian Matthias; Stevenson, Keith J.; Webb, Lauren JWhile previous work has demonstrated the effectiveness of nitrogen-doped carbon nanotubes (N-CNTs) as biogenic electrode materials in first- and second-generation biosensors, this thesis primarily explores enzymatic inhibition-based biosensing schemes on N-CNT electrodes. This type of scheme enables the detection of enzymatic inhibitors, as opposed to enzymatic substrates, making these inhibition-based biosensors much more suitable for the monitoring of environmental pollutants. Presented in this thesis is a biosensor which couples N-CNTs with glucose oxidase (GOx) through spontaneous physical adsorption for the highly sensitive detection of aqueous silver ions. Included is a thorough discussion of the parameters that affect response time as well the biosensor’s aptitude for repeated use. A later chapter presents initial work towards the inhibition-based detection of sucralose, a relatively new environmental pollutant. A bi-enzymatic approach is explored, in which both GOx and invertase are immobilized on an N-CNT modified electrode. Finally, shifting focus from inhibition-schemes, the last remaining chapter investigates the coupling of CNTs and N-CNTs with methylene green (MG), a redox mediator used in second-generation biosensors based on NADH oxidation. Common coupling techniques are examined for their effectiveness in decreasing the overpotential required for NADH oxidation.