Development of isotope labeling liquid chromatography mass spectrometry for metabolome analysis

  • Author / Creator
    Guo, Kun
  • As the endpoint of the “omics” cascade, metabolomics has attracted much attention in the discovery of diagnostic or prognostic disease biomarkers, and as a powerful tool to understand biological and biochemical processes and mechanisms. The objective of my research was to develop a highly sensitive and reliable method for quantitative and qualitative metabolome analysis, based on a global 13C-/12C-stable isotope labeled internal standards (SIL IS) strategy and reversed-phase liquid chromatography (RPLC) Fourier Transform Ion Cyclotron Resonance (FT ICR) Mass Spectrometry (MS). In conventional LC-MS-based metabolome analysis, quantification is mainly based on the peak intensities, whilst qualification is based on accurate mass measurement. The SIL IS strategy, which provides isotopic labeled internal standards for every targeted analyte, has been proved to be the most effective approach to overcome ion suppression and matrix effects. Thus, it offers the most accurate quantification and confident identification. This thesis has focused on four aspects of current metabolomics research: (1) development of chemical derivatization chemistries to improve electrospray ionization (ESI) response and reversed-phase liquid chromatography separation of human metabolites, with particular focus on polar, and/or non-ESI ionizable metabolites; (2) development of a novel 13C-/12C- differential isotope labeling (DIL) strategy for accurate quantification and confident identification of human metabolites; (3) development of a software tool for DIL quantification and putative and definitive identification; and (4) construction of a comprehensive, targeted metabolite library. 13C-/12C-dansylation targeted primary and secondary amines, and phenolic hydroxyl metabolites. An isotope labeling method, based on the use of 13C-/12C-isotope-coded p-dimethylaminophenacyl (DmPA) bromide as a reagent, targeted carboxylic acid-containing metabolites (CAMs). Both 13C-/12C-labeling methods offered several desirable features, including simple and robust experimental procedures, no isotopic effects on reversed-phase separations, significant ESI enhancement, and improvement of the reversed-phase separation. The construction of a comprehensive 13C-/12C-labeled metabolite library and software tool ensured that accurate quantification and confident identification of metabolites could be carried out in a high-throughput, automatic fashion. The DIL strategy is a global internal approach that could be applied to many other LC-MS applications beyond metabolome analysis.

  • Subjects / Keywords
  • Graduation date
    Spring 2011
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • 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
  • Institution
    University of Alberta
  • Degree level
  • Department
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Harrison, Jed (Department of Chemistry)
    • Agnes, George (Department of Chemistry, Simon Fraser University)
    • Lucy, Charles (Department of Chemistry)
    • Li, Liang (Department of Chemistry)
    • Clive, Derrick (Department of Chemistry)
    • Bamforth, Fiona (Department of Laboratory Medicine and Pathology)