Example of full 1H-NMR data set

To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum 'Moneymaker') plants were grown under contrasting conditions (optimal for commercial, shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids.

For more details, see FRIM - Fruit Integrative Modelling, an ERASysBio+ projet, and the references given below.

The available dataset for download can be simply depicted and summarized by the following figure:

References

Bénard C., Bernillon S., Biais B., Osorio S., Maucourt M., Ballias P., Deborde C., Colombié S., Cabasson C., Jacob D., Vercambre G., Gautier H., Rolin D., Génard M., Fernie A., Gibon Y., Moing A. 2015 Metabolomic profiling in tomato reveals diel compositional changes in fruit affected by source-sink relationships. Journal of Experimental Botany Vol. 66, No. 11 pp. 3391–3404 doi:10.1093/jxb/erv151

Beauvoit B., Colombié S., Monier A., Andrieu M.-H., Biais B., Bénard C., Chéniclet C., Dieuaide-Noubhani M., Nazaret C., Mazat J.-P., Gibon Y. (2014). Model-assisted analysis of sugar metabolism throughout tomato fruit development reveals enzyme and carrier properties in relation to vacuole expansion. Plant Cell 26: 3224–3242

Biais B, Bénard C, Beauvoit B, Colombié S, Prodhomme D, Ménard G, Bernillon S, Gehl B, Gautier H, Ballias P, Mazat J-P, Sweetlove L, Génard M, Gibon Y. 2014. Remarkable reproducibility of enzyme activity profiles in tomato fruits grown under contrasting environments provides a roadmap for studies of fruit metabolism. Plant Physiology 164, 1204-1221.

Colombié S, Nazaret, Bénard C, Biais B, Mengin V, Solé M, Fouillen L, Dieuaide- Noubhani M, Mazat J, Beauvoit B, Gibon Y. 2015. Modelling central metabolic fluxes by constraint-based optimization reveals metabolic reprogramming of developing tomato fruit. The Plant Journal 81, 24-39.

For 1H-NMR profiling, polar metabolites, were extracted from the ground lyophilised samples as previously described (Biais et al., 2009; Moing et al., 2004) with minor modifications. Frozen powdered samples were lyophilised and polar metabolites were extracted from 20 mg of lyophilised powder with an ethanol-water series at 80°C. The supernatants were combined, dried under vacuum and lyophilised. Two technical replicates were prepared for each fruit sample. Each lyophilised extract was solubilised in 500 µL of 250 mM potassium phosphate buffer solution at apparent pH 6.0, 5 mM ethylene diamine tetraacetic acid disodium salt (EDTA), in D2O, titrated with KOD solution to pH 6.00 ±0.02 when necessary, and lyophilised again. The lyophilised titrated extracts were stored in darkness under vacuum at room temperature, before 1H-NMR analysis was completed within one week.

Before 1H-NMR analysis, 500 µL of D2O with sodium trimethylsilyl [2,2,3,3-d4] propionate (TSP, 0.01% final concentration for chemical shift calibration) were added to the lyophilised titrated extracts. The mixture was centrifuged at 10,000 g for 5 min at room temperature. The supernatant was then transferred 1 into a 5 mm NMR tube for acquisition. Quantitative 1H-NMR spectra were recorded at 500.162 MHz and 300 K on a Bruker Avance III spectrometer (Wissembourg, France) using a 5-mm broadband inverse probe, a 90° pulse angle and an electronic reference for quantification (Biais et al., 2009; Mounet et al., 2007).

The assignments of metabolites in the NMR spectra were made by comparing the proton chemical shifts with literature (Fan, 1996; Mounet et al., 2007) or database values (MeRy-B, HMDB), by comparison with spectra of authentic compounds recorded in the same solvent conditions (in-house library) and by spiking the samples. 1H-1H COSY NMR experiments were acquired for selected samples for assignment verification.

For absolute quantification of metabolites, four calibration curves (glucose and fructose: 1.25 to 50 mM, glutamate and glutamine: 0 to 15 mM) were prepared and analysed under the same conditions. The glucose calibration was used for the absolute quantification of all compounds, as a function of the number of protons of selected resonances except fructose, glutamate and glutamine quantified using their respective calibration curve.

References

Biais B, Allwood JW, Deborde C, Xu Y, Maucourt M, Beauvoit B, Dunn WB, Jacob D, Goodacre R, Rolin D, Moing A. 2009. H-1 NMR, GC-EI-TOFMS, and Data Set Correlation for Fruit Metabolomics: Application to Spatial Metabolite Analysis in Melon. Analytical Chemistry 81, 2884-2894.

Fan TWM. 1996. Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures. Progress in Nuclear Magnetic Resonance Spectroscopy 28, 161- 219.

Moing A, Maucourt M, Renaud C, Gaudillere M, Brouquisse R, Lebouteiller B, Gousset-Dupont A, Vidal J, Granot D, Denoyes-Rothan B, Lerceteau-Kohler E, Rolin D. 2004. Quantitative metabolic profiling by 1-dimensional H-1-NMR analyses: application to plant genetics and functional genomics. Functional Plant Biology 31, 889-902.

Mounet F, Lemaire-Chamley M, Maucourt M, Cabasson C, Giraudel JL, Deborde C, Lessire R, Gallusci P, Bertrand A, Gaudillere M, Rothan C, Rolin D, Moing A. 2007. Quantitative metabolic profiles of tomato flesh and seeds during fruit development: complementary analysis with ANN and PCA. Metabolomics 3, 273-288.

Input files

Calibration files

Output files

Step 1: Load the dataset

• Download files in the 'input files' list, namely: 'NMRFRIM3-4.zip', 'samples_p1.txt' and 'NP_macro_cmd_NMRFRIM3-4.txt'
• Start NMRProcFlow from your web browser.
• Choose 'Bruker' as Instrument/Vendor/Format, and '1r spectrum' as Spectra type.
• Then load the ZIP file and the sample file as described in the 'Data preparation phase' section
• Before launchning, you have also to load the macro-command file in the form for advanced user as described in the 'Replay the same processing workflow' section
• Finaly, click on the 'Launch' button'

Step 2: Import the bucket table

• Download the bucket table file 'buckets_FRIM3-4.txt'.
• Import the bucket table as described in the 'Bucketing' section. Don't forget to select the 'Tabular Separator Value' as the import format.

Step 3: Export the XLSX workbook for quantification

• Download the XLSX workbook file 'wb_NMRFRIM3-4.xlsx'.
• Export the XLSX workbook for quantification as described in the 'Data Export' section