Metabolomics is a rapidly growing approach based on global analysis of all metabolites in a tissue, fluid or organism. This approach is based on the assumption that the metabolome serves as a holistic measure of metabolic status. Most metabolomic analyses have been based on bulk sample analyses, which do not differentiate metabolites from specific tissues or cells. Since metabolic activity is organized in complex networks, bulk analyses involve a loss of potentially important chemical information. This dissertation presents development of analytical methods to profile localized metabolites in biological tissues, and applies these to investigations of glandular trichomes, which are epidermal plant cells that are prolific at chemical synthesis. Microsampling of individual trichomes from wild tomato relatives of the genus Solanum was achieved using micropipettes, and blasting of tissue from surfaces of corals was performed using a waterpik. Metabolite detection, identification, and quantification were performed using liquid chromatography/mass spectrometry (LC/MS). Through accurate mass measurements and chromatographic peak alignments, 109 metabolite signals were annotated from extracts of single trichome types. Methylated flavonoids represented one class of trichome metabolites, but mass spectra alone were not adequate to distinguish isomers. Complete assignment of isomeric methylated myricetins was achieved by employing two recombinant O-methyltransferase enzymes from Solanum habrochaites LA1777 for selective semisynthesis of methylated myricetins. Tandem mass spectrometric analysis and chromatographic retention times provided evidence needed for structure annotation, aided by selective enzymatic incorporation of deuterium labeled methyl groups. These structural assignments aided demonstration of substrate selectivity of the two methyltransferases. To accelerate profiling of individual trichomes, a contact printing approach was developed to transfer cells from across an entire leaf to a carbon substrate while preserving spatial information during the transfer. Direct and matrix-free laser desorption ionization from the printed cells allowed chemical imaging of trichomes across an entire leaflet. Individual trichome resolution was achieved using smaller steps of stage position, yielding about 50 ìm resolution. Using this scheme, the first spatially-resolve profiling of trichome metabolites across the Solanum leaflet was achieved, and demonstrated chemical heterogeneity among trichomes within a single type. Localized metabolite profiling was also performed for reef coral tissues collected from a field location near Curacao. A stable isotope dilution method was developed for quantifying seven betaine metabolites, which may serve as photosystem stabilizing substances, and dimethylsulfoniopropionate (DMSP) in extracts of coral tissues. The LC-MS protocol employed a pentafluorophenylpropyl (PFPP) column and time-of-flight mass spectrometry. The results demonstrate that corals accumulate multiple betaines, that betaine profiles vary widely among different coral species, and that betaine levels are correlated with ambient light levels.
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