Thesis
Metabonomic analysis of Drosophila mutants using high resolution mass spectrometry
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2013
- Thesis identifier
- T13317
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- The availability of fully sequenced genomes of model organisms such as Drosophila and their subsequent annotation has afforded opportunities for reverse genetics in a complex model organism. Metabonomics used as an aid to functional genomics can be used to understand the functions of genes in living systems. Thus metabonomics has been employed to study Drosophila samples extracted from whole animals at different developmental stages or in response to external stimuli or genetic mutation. With unmatched mass resolution, accuracy, and detection sensitivity, linear ion trap - Fourier Transform Orbitrap Mass Spectrometry (LTQ-Orbitrap-MS) has the potential for high throughput metabonomic analysis. Five different but linked studies are reported in this work. A global metabolic profiling method based on electrospray ionisation mass spectrometry was developed for Drosophila melanogaster metabolites. The method involved optimizing the extraction of Drosophila metabolites followed by analysis using liquid chromatography coupled with high-resolution mass spectrometry. The effect of extraction conditions and storage were studied, thus 750-800 metabolites were putatively identified in order to obtain the metabolite profiles of Drosophila reference strains and mutants. Metabolic studies were carried out to elucidate gene functions using established protocols. The online resource FlyAtlas.org provides detailed microarray-based expression data for the tissues and life-stages of Drosophila. Since downstream genes, such as urate oxidase are tubule-specific, an Orbitrap technology has been used to elucidate tissue specific metabolomes. Additionally, genetic interventions using designed RNA interference were also made and validated by qPCR and metabonomics. The method produced a new opportunity for metabonomics use in validating gene expressions. The xanthine oxidase inhibitor allopurinol was used to phenocopy the rosy mutation which caused the levels of xanthine and hypoxanthine to rise while the levels of uric acid fell. In addition, many unexpected metabolic changes followed this treatment with effects on the pentose phosphate pathway and tryptophan metabolism being the most marked. The yellow (y) gene was first discovered in Drosophila, but occurs in many insect species and in some bacteria. The y protein is similar to the major royal jelly proteins produced by bees. Metabolomic profiling was carried out on Oregon R (OR) and y Drosophila larvae at the third instar. There were numerous metabolic differences between the metabolic profiles of OR and y. Phenylalanine, tyrosine and DOPA were all elevated in y, as might be expected since the mutation blocks melanin biosynthesis. In addition, there were other metabolic effects including marked effects on to lysine metabolism. The white mutation of Drosophila, which affects ABC transporters, was studied with regards to its effect on pigment biosynthesis in Drosophila. In addition to the expected effects on pigments there were interesting male/female differences possibly related to the presence of the white gene in the X chromosome. In addition the effect of salt stess on wild type and white flies was studied. Overall, LTQ-Orbitrap-MS proved suitable for metabonomic analysis of both wild-type and mutant Drosophila and had potential in the analysis of metabolomes of single tissues. The possibility of using Orbitrap-based metabonomics in combination with Drosphila for drug testing is discussed and is a goal for the future.
- Resource Type
- DOI
- Date Created
- 2013
- Former identifier
- 967041
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