Bioanalytical applications of mass spectrometry
Warren, Maria Rosario Esteban
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MALDI and ESI mass spectrometry have been invaluable techniques for the analysis of a wide range of biomolecules including peptides, proteins, carbohydrates and glycoproteins. Although used extensively, the relatively new techniques still require improvements in the sample preparation process. These improvements are necessary to enhance the efficiencies and broaden the applications of MALDI and ESI towards problematic biomolecular samples. In addition, existing analytical techniques should be evaluated, modified and applied to study different biological systems. The scope of this dissertation involves an improvement to peptide and protein analysis by MALDI -MS and application of existing techniques of MALDI and ESI for studies involving a fungal enzyme. Biomolecular samples can contain a variety of compounds, which at moderate or elevated levels interfere with their analysis by MALDI-MS. MALDI sample probes modified with self-assembled monolayers containing ionic terminal groups were developed and evaluated as an on-line purification or sample-cleansing device. Various types of probes were successful in removing several types of common buffer components or contaminants. These probes also exhibit useful characteristics for routine analysis. MALDI and ESI- LC-MS were also used in studies of recombinant forms of Aspergillus niger pectin methylesterase. The specificity of the enzyme towards substrates, which contained multiple sites of potential activity was investigated. The specificity was determined by structural analysis of the products formed as a result of the enzymatic activity of pectin methylesterase. Furthermore the enzyme itself is a glycoprotein. The structures and location of these carbohydrates were determined primarily using LC-ESIMS as a carbohydrate-specific detection technique.