Inter-laboratory reproducibility of fast gas chromatography–electron impact–time of flight mass spectrometry (GC–EI–TOF/MS) based plant metabolomics

Abstract

The application of gas chromatography–mass spectrometry (GC–MS) to the ‘global’ analysis of metabolites in complex samples (i.e. metabolomics) has now become routine. The generation of these data-rich profiles demands new strategies in data mining and standardisation of experimental and reporting aspects across laboratories. As part of the META-PHOR project’s (METAbolomics for Plants Health and OutReach: http://www.meta-phor.eu/) priorities towards robust technology development, a GC–MS ring experiment based upon three complex matrices (melon, broccoli and rice) was launched. All sample preparation, data processing, multivariate analyses and comparisons of major metabolite features followed standardised protocols, identical models of GC (Agilent 6890N) and TOF/MS (Leco Pegasus III) were also employed. In addition comprehensive GC×GC–TOF/MS was compared with 1 dimensional GC–TOF/MS. Comparisons of the paired data from the various laboratories were made with a single data processing and analysis method providing an unbiased assessment of analytical method variants and inter-laboratory reproducibility. A range of processing and statistical methods were also assessed with a single exemplary dataset revealing near equal performance between them. Further investigations of long-term reproducibility are required, though the future generation of global and valid metabolomics databases offers much promise.

Acclimation of metabolism to light in Arabidopsis thaliana: the glucose 6‐phosphate/phosphate translocator GPT2 directs metabolic acclimation

“…Tissue aliquots (30 mg DW) were taken for the GC‐EI‐TOF/MS extraction procedure, which precisely followed that of Allwood et al . (2009) and Lisec et al . (2006).…”

Transcriptomic and Metabolic Changes Associated with Photorespiratory Ammonium Accumulation in the Model Legume Lotus japonicus

“…Metabolite profiles were analyzed with the TagFinder software (Luedemann et al, 2008) and filtered for those metabolic features represented by three or more intercorrelated mass fragments within each independent experiment. The validity of this analytical approach to quantify metabolites in plant tissues has been demonstrated (Allwood et al, 2009). The resulting data were normalized to internal standard (a mixture of ribitol, 2,3,3,3-d 4 -DL-Ala, and D[2]-isoascorbic acid) and FW.…”

FUM2, a Cytosolic Fumarase, Is Essential for Acclimation to Low Temperature in Arabidopsis thaliana

“…Briefly, flash-frozen leaves were lyophilized and stored at 280°C. Tissue (30 mg dry weight) was extracted into methanol/water as described previously (Lisec et al, 2006;Allwood et al, 2009). Polar phase extracts were dried and spiked with 100 mL of an internal standard solution containing 200 mg/mL succinic-d 4 acid, Gly-d 5 , and malonic-d 2 acid before analysis.…”