Poster: Integrated Plant Biology
Abs #
85: Simultaneous profiling of amino acids and both reduced and oxidized forms of glutathione, cysteine, and others thiols during Arabidopsis growth and development
|
|
Presenter: |
Martin, Melinda N., mnmartin@aesop.rutgers.edu |
Authors | Martin, Melinda N. (A) Leustek, Thomas (A) | | Affiliations: |
(A): Rutgers Univeristy
|
|
|
Cysteine is the precursor for all molecules containing reduced sulfur (-2 valance). These include methionine, the tripeptide glutathione (GSH, g-Glu-Cys-Gly) and their many metabolites. To analyze the impact of genetically manipulating steps in the anabolic and catabolic pathways of Cys, Met, and GSH a single facile profiling method was needed. Although flux through these pathways is high, the tissue level of many soluble metabolites is very low. Metabolites with thiol groups (-SH) such as GSH and Cys present unique challenges including that they are also present in plant tissues as a disulfide (S-S) which can readily form by oxidation during sample handling. We have used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) to simultaneously tag both amine and thiol groups for fluorescence detection and have resolved the adducts by high performance liquid chromatography interfaced with a fluorescence detector. Cohen & Michaud (Anal Biochem 1993 211, 279-287) first described the use of AQC for analysis of amino acids in protein hydrolysates. We modified the reaction so as to form stable adducts with thiol compounds and designed chromatographic conditions so as to resolve 80 to 100 metabolites including major amino acids, many intermediates, and thiols including Cys, GSH, g-Glu-Cys, Cys-Gly and their disulfide forms. AQC offers significant advantages over other methods for the tagging of thiols and/or amino acids. The soluble components in crude plant extracts can be derivatized without sample cleanup. The reaction occurs in seconds, the adducts are stable, and no clean up is required for chromatography. Sensitivity is in the fmol to low pmol range permitting us to profile amino acids and thiols in minuscule samples such as the parts of a single Arabidopsis flower.
