Improving the Quantity and Quality of Plant Metabolomics Information
Plants to be Studied: Arabidopsis, grape vine, tomato, and rice
Project Objectives: Metabolomics methodology is fundamentally limited by multiple analytical challenges including: 1) metabolite identification (annotation), 2) quantification, 3) spatial localization, 4) detection over a large dynamic range, and 5) measurement of metabolic flux. We propose to generate new strategies and community resources to begin to address the challenges associated with compound identification and quantification through the following objectives:
1) Provide improved sample preparation and derivatization methods and create new spectral library resources for metabolite identification by LC- and GC-MS.
2) Use stable isotope metabolically labeled plant materials for isotope dilution with relative and absolute quantification methods by GC-MS/MS and LC-MS/MS.
Experimental Approaches: We will develop strategies for automated solid phase extraction and derivatization of metabolites that increase the numbers of observable compounds in both GC- and LC-MS and provide complementary information about the functional groups on the compounds as an aid to compound structural elucidation. We also plan to evaluate variables associated with tandem MS across multiple platforms that contribute to differences in fragmentation patterns in order to understand how these differences may be minimized or understood. In this way community based MS/MS library information will be rendered more generally useful. We will generate and disseminate GC-MS and -MS/MS and LC-MS/MS standard metabolite spectral libraries that include metabolites derivatized using the strategies developed above.
We will produce 13C labeled tomato and rice tissues for isotope dilution quantification standards by growing plants from seed in a controlled 13CO2 atmospheric chamber. These materials will be used to develop absolute and relative methods and computational tools for quantification by MS. We will also evaluate other methodologies for stable isotope incorporation into complex plant metabolite populations using chemical means when metabolic labeling is not feasible.