Development and Application of Isotope Labeling Method and Data Processing Program for Liquid Chromatography Mass Spectrometry Based Metabolome Profiling

Abstract

The objective of this thesis focuses on establishing a software tool (IsoMS) used in the quantification and qualification of metabolites in complex biological samples, developing robust methods to extract metabolites from different plant samples, and applying a differential 13C-/12C-isotope dansylation labeling technique with liquid chromatography-mass spectrometry (LC-MS) for the analysis of amine- and phenol-containing metabolites in plant and human urine samples. This approach, a differential 13C-/12C-isotope dansylation labeling method combined with LC-MS, has successfully been applied to the relative quantification, peak pair identification, and putative metabolite identification in a study of metabolic profiling. First of all, IsoM has been developed to handle the raw LC-MS data generated by a chemical isotope labeling or isotope coded derivatization (ICD) metabolomics platform by peak picking, peak pairing, peak-pair filtering and peak-pair intensity ratio calculation. Secondly, a protocol for rapid extraction, derivatization and detection of amine- and phenol-containing metabolites in mg-scale samples of flax (Linum usitiatissimum) bast fibers has been developed and can typically be completed in under 13 hours for up to 8 samples. Thirdly, the development of a robust metabolite extraction method from ginseng roots tailored to the analysis of amine- and phenol-containing metabolites and the use of the dansylation labeling method to gauge the detectability of the LC-MS technique for the ginseng metabolome has been used on the application of spatial distribution of metabolites in ginseng roots. Fourthly, a microwave-assisted extraction method of Arabidopsis thaliana has been optimized and used prior to a differential 13C-/12C-isotope dansylation labeling technique with LC-FT-MS for the study of metabolic profiling of different TIFY gene expression plants under methyl jasmonate treatment. Finally, a highly sensitive dansylation isotope labeling LC-MS method was used to determine the urine metabolomes before and after a moderate amount of drinking Goji tea, considered as a phyto-nutritional supplement drink. The result clearly showed that the consumption of Goji tea does not affect the urine metabolome significantly for the studies of the short term (<3 hr) and longer term (12 hr) effects of drinking Goji tea.