Simultaneous analysis of metabolites in potato tuber by gas chromatography–mass spectrometry

Abstract

SummaryA new method is presented in which gas chromatography coupled to mass spectrometry (GC±MS) allows the quantitative and qualitative detection of more than 150 compounds within a potato tuber, in a highly sensitive and speci®c manner. In contrast to other methods developed for metabolite analysis in plant systems, this method represents an unbiased and open approach that allows the detection of unexpected changes in metabolite levels. Although the method represents a compromise for a wide range of metabolites in terms of extraction, chemical modi®cation and GC±MS analysis, for 25 metabolites analysed in detail the recoveries were found to be within the generally accepted range of 70±140%. Further, the reproducibility of the method was high: the error occurring in the analysis procedures was found to be less than 6% for 30 out of 33 compounds tested. Biological variability exceeded the systematic error of the analysis by a factor of up to 10. The method is also suited for upscaling, potentially allowing the simultaneous analysis of a large number of samples. As a ®rst example this method has been applied to soil-and in vitro-grown tubers. Due to the simultaneous analysis of a wide range of metabolites it was immediately apparent that these systems differ signi®cantly in their metabolism. Furthermore, the parallel insight into many pathways allows some conclusions to be drawn about the underlying physiological differences between both tuber systems. As a second example, transgenic lines modi®ed in sucrose catabolism or starch synthesis were analysed. This example illustrates the power of an unbiased approach to detecting unexpected changes in transgenic lines.

Reduced Expression of Aconitase Results in an Enhanced Rate of Photosynthesis and Marked Shifts in Carbon Partitioning in Illuminated Leaves of Wild Species Tomato

“…Data are presented normalized to wild type as detailed by Roessner et al (2001b). The recovery of small, representative amounts of each metabolite through the extraction, storage, and assay procedures of the chromatography methods has been documented previously (Roessner et al, 2000;Fernie et al, 2001b). Chlorophyll was measured in 80% (w/v) acetone extracts as described by Lichtenthaler (1987).…”

Metabolomics — the link between genotypes and phenotypes

“…Leaf tissues, for example, can be ground under liquid nitrogen simply using mortar and pestle, or using a ball mill with pre-chilled holders (Fiehn et al, 2000a), or together with the extraction solvent by ultraturrax devices (Orth et al, 1999). Other plant organs such as roots, however, prove sometimes to be too hard to use for ball mills, whereas potato tubers are too soft (Roessner et al, 2000). After homogenization, different methods of metabolite extraction could be used but, again, no systematic study is available that directly compares the results of these techniques.…”

“…Derivatization conditions for metabolite profiling were optimized by selecting 12 compounds representative of plant primary metabolism (Adams et al, 1999), and were applied to profile polar organics (sugars, polyols, acids and amino acids) in apricots (Katona et al, 1999). These techniques were used more systematically to analyse the number of metabolites involved in potato tuber primary metabolism by evaluating recovery rates using spiking experiments and external calibration curves (Roessner et al, 2000). Another step in elucidating metabolism was taken by Christensen and Nielsen (2000), who used GC/MS to profile the fractional enrichment of 13 C-labelled substrates in order to study biochemical pathways.…”

Global metabolite analysis of yeast: evaluation of sample preparation methods

“…Sugars, sugar phosphates, sugar alcohols and peptides were determined simultaneously by GC-MS after methoxymation and silylation derivatization, following the protocol described by Roessner et al (2000). The determination of extracellular sugar phosphates for the evaluation of leakage during quenching was performed using solid-phase extraction and high-pressure ion-exchange chromatography with pulsed amperometric detection (HPIC-PAD), according to Smits et al (1998).…”