Comparative Analysis of Amino Acids from Arabidopsis Wild-Type and Mutant sir1-1 Leaves by Reverse-Phase High-Pressure Liquid Chromatography (HPLC) and Gas Chromatography-Mass Spectrometry (GC-MS)

The accepted gold standard for determination of amino acids levels in eukaryotic cells is derivatization of extracted amino acids with a fluorescent dye followed by separation of the respective derivates by high performance liquid chromatography (HPLC). The method is highly sensitive but has a narrow spectrum of metabolites that can be analyzed. The coupling of gas chromatography to a sensitive mass-spectrometer (GC-MS) allows analyzing a wide range of metabolites in one run. In this study both methods for determination of amino acid levels were compared in wild-type and sir1-1 an Arabidopsis thaliana T-DNA knock in mutant in the promoter of sulfite reductase. Sulfite reductase is a key enzyme in the sulfate reduction pathway of plants and is exclusively localized in plastids. Both methods confirm independently a significant increase of total amino acids level in leaves of sir1-1 plants in comparison to the wild-type. The degree of increase for each amino acid detected by GC-MS could be confirmed in almost all cases by HPLC. The GC-MS method is suitable for screening of mutants disturbed in amino acid composition and the assessment of adaptations in primary metabolism of various mutants.