Analytical strategies for profiling, annotation, and structure elucidation of specialized terpenoid metabolites
The main bottleneck in today's plant metabolomics lies with the identification of new metabolites. A number of plant metabolite databases that report liquid chromatography - mass spectrometry data have been constructed. However, more than 95% of compounds reported in these databases remain unannotated. The extensive range of unknown metabolites presents a significant challenge in interpreting metabolome data, and therefore developing methods that accelerate annotation and identification of previously unknown metabolites has great importance when metabolome data are used for functional genomic research. The challenge of metabolite annotation was addressed by using relative mass defect (RMD) filtering of ion masses measured using liquid chromatography-mass spectrometry. Such calculated RMD values reflect the fractional hydrogen content of each detected ion, and reflect the biosynthetic precursors and transformations that generate metabolites in vivo. RMD filtering aids grouping of compounds of similar relative mass defect independent of absolute mass and chromatographic retention time. Therefore, metabolites and metabolite precursors are grouped together enabling potential associations among related metabolites to be developed. Furthermore, a systematic variation of RMD among the fragment/product ions observed in multiplexed collision-induced dissociation (CID) MS or liquid chromatography-tandem mass spectrometry (LC-MS/MS) data for compounds of interest allowed for the identification of terpene glycosides in complex matrices. However, once the metabolites are annotated in metabolomics data sets, establishing the structure of these compounds requires the purification of the compound followed by de novo structure elucidation that relies heavily upon 1D and 2D NMR.Chapter 2 of this dissertation discusses the application of RMD filtering based data analysis to both parent and fragment ions generated in LC-multiplexed CID MS metabolite profiles generated from wild tomato species Solanum habrochaites LA1777. This resulted in the discovery of over 24 novel sesquiterpene glycoside chemical formulas, with multiple isomers comprising a group of more than 200 sesquiterpenoid glycosides. Chapters 3 and 4 of this dissertation discuss the purification and de novo structure elucidation of seven example compounds from wild tomato glandular trichomes using NMR. The structures of the sesquiterpenoid cores established for these compounds are different from the structures of known volatile sesquiterpenoid compounds found in S. habrochaites LA1777 suggesting that the synthesis of these non-volatile terpenoids involves different biosynthetic enzymes from those involved in the synthesis of known volatile terpenoids. Similarly, Chapter 5 of this dissertation discusses the application of these techniques to the analysis of metabolite profiles of the medicinal plant Hoodia gordonii generated using LC-multiplexed CID MS. This research led to the identification of 24 novel diterpene glycosides. These compounds are believed to share the diterpenoid cores found in some of the known diterpene glycosides from Hoodia gordonii and therefore these compounds are likely biosynthetic intermediates of the synthesis of some of the known diterpene glycosides.
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- In Collections
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Electronic Theses & Dissertations
- Copyright Status
- In Copyright
- Material Type
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Theses
- Authors
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Ekanayaka, Prabodha
- Thesis Advisors
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Jones, Arthur D.
- Committee Members
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Blanchard, Gary
Spence, Dana
Walker, Kevin
- Date
- 2014
- Program of Study
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Chemistry - Doctor of Philosophy
- Degree Level
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Doctoral
- Language
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English
- Pages
- xvi, 194 pages
- ISBN
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9781321103052
1321103050
- Permalink
- https://doi.org/doi:10.25335/hmwv-3k28