Targeted metabolomics on the shikimate and aromatic amino acid biosynthetic pathways

Wheeler, Robin E. M.S., Purdue University, May 2016. Targeted Metabolomics on the Shikimate and Aromatic Amino Acid Biosynthesis Pathways. Major Professor: John A. Morgan. The shikimate and aromatic amino acid biosynthesis pathways are some of the most studied biosynthetic pathways in nature due to their fundamental importance. Interest in this field stems from synthesis of the essential amino acids, tryptophan, tyrosine and phenylalanine. The pathways also are important as generating the precursors of thousands of secondary metabolites and lignin. Inhibiting the pathway has led to discovery of herbicides, pesticides as well as tuberculosis drugs. However there is currently a lack of a comprehensive targeted method for detecting and quantifying the majority of the intermediates in these pathways. Often multiple extraction and detection methods are used in a single paper to gather the desired data, which are a strain on time and resources. Here we developed two more comprehensive methods. The methods consisted of a single extraction coupled with an acidic and a neutral acetate buffer Using TBA as an ion paring agent. The chromatography was in the reverse phase mode on a C8 column. In the neutral extracts we used OPA as a derivitization reagent, while in the acidic method the extracts were run directly after concentration. The compounds are further separated and quantified in a MS/MS with a triple-quad detector. We are able to separate and detect 15 compounds in standards and validated the method on plant extracts from Arabidopsis