The work presented herein is a method optimization for biomolecule detection and identification using Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI). MALDI-MSI is a unique form of mass spectrometry that is highly multiplexed; it can simultaneously retain location information of the mass of multiple ions, allowing for correlation of morphology or pathology to reconstructed ion heat maps. There were three main objectives for the research - 1) A method optimization of sample preparation techniques for bottom-up proteomic MALDI-MSI was performed. This included the optimization of tissue wash steps, trypsin digestion incubation times, and matrix deposition techniques. The results included identifying the appropriate pH for the wash steps to optimize trypsin digestion, an overnight trypsin incubation to allow for complete digestion, and the inclusion of MCAEF – Matrix Coating Assisted by an Electric Field – during matrix coating for enhanced spectra. 2) An unbiased statistical data processing workflow for simultaneous processing of multiple datasets was performed. This was done using a thyroid hormone treated tadpole dataset to gain insight into the metabolism of anuran metamorphosis. Results found included a finalized data processing workflow that detected 5000 metabolite features from five organs were detected in pre-metamorphic tadpoles. Of these detected metabolites, 136 were significantly affected upon exposure to thyroid hormone and 64 metabolites were putatively identified. 3) A sample preparation technique for metabolomic analysis of formalin-fixed paraffin embedded (FFPE) colorectal liver metastasis samples was performed. Results included the importance of using a high mass resolution mass spectrometer while emphasizing more appropriate use of fresh-frozen tissue sections for metabolomic analysis. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/8913 |
| Date | 22 December 2017 |
| Creators | Luehr, Teesha Crystal |
| Contributors | Borchers, Christoph H. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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