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Metabolite Identification and Quantification in Biofluids Using Liquid Chromatography Mass Spectrometry

  • Author / Creator
    Stanislaus, Avalyn E
  • Metabolomics, one of the branches of systems biology, is the comprehensive measurement of all the endogenous metabolites in a biological system. It can be applied in the areas of biomarker discovery and diagnosis of critical diseases and disorders, and can significantly increase our understanding of the pathophysiology involved in development of acute diseases. For metabolomics to reach its true potential, there are many key areas that need to be addressed. This thesis focuses on the development and application of new mass spectrometric techniques aimed at improving several areas in metabolomics research: 1) detection and identification of compound classes, 2) enhancing the electrospray response and improving the chromatographic behavior of metabolites, 3) increasing sample throughput, 4) accurate quantification of endogenous metabolites and 5) expanding current databases used in metabolite identification. The initial work, the comprehensive analysis of acylglycines in human urine, illustrates the analytical challenges of detection and identification of trace levels of metabolites in urine. Mass spectrometric methods were optimized using fragmentation patterns and breakdown graphs, and putative identifications were made using a combination of diagnostic neutral losses. This method was successful in detecting and putatively identifying 43 new acylglycines. Quantification of these metabolites in human urine and plasma was performed using a new labeling strategy. Chemical modification was advantageous as it enhanced the electrospray response of these polar metabolites and provided a way to incorporate a stable isotope onto the molecule. Quantification using a new “surrogate matrix” strategy was also developed. A new fast LC method was developed for the analysis of several analytes in the vitamin B12 pathway in less than 36 seconds. This method was very useful in improving high-throughput and in the discovery of the role of ABC protein transporters in B12 metabolism. Finally, a web-based software called MyCompoundID was developed to expand the metabolome coverage of an existing database (HMBD) by generating metabolic products derived from 76 common biotransformations. This served to increase the number of entries from 8000 to more than 10 million.

  • Subjects / Keywords
  • Graduation date
    2012-11
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R39W0970G
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Chemistry
  • Supervisor / co-supervisor and their department(s)
    • Li, Liang (Chemistry)
  • Examining committee members and their departments
    • Hall, Dennis (Chemistry)
    • Harynuk, James (Chemistry)
    • Waldron, Karen (Chemistry)
    • Klassen, John (Chemistry)
    • Curtis, Jonathan (Agricultural, Food and Nutritional Science)