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1H NMR metabolite fingerprinting and metabolomic analysis of perchloric acid extracts from plant tissues

Abstract

Metabolite fingerprinting provides a powerful method for discriminating between biological samples on the basis of differences in metabolism caused by such factors as growth conditions, developmental stage or genotype. This protocol describes a technique for acquiring metabolite fingerprints from samples of plant origin. The preferred method involves freezing the tissue rapidly to stop metabolism, extracting soluble metabolites using perchloric acid (HClO4) and then obtaining a fingerprint of the metabolic composition of the sample using 1D 1H NMR spectroscopy. The spectral fingerprints of multiple samples may be analyzed using either unsupervised or supervised multivariate statistical methods, and these approaches are illustrated with data obtained from the developing seeds of two genotypes of sunflower (Helianthus annuus). Preparation of plant extracts for analysis takes 2–3 d, but multiple samples can be processed in parallel and subsequent acquisition of NMR spectra takes 30 min per sample, allowing 24–48 samples to be analyzed in a week.

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Figure 1: Representative 600-MHz 1H NMR spectrum of a perchloric acid extract of developing sunflower embryos.
Figure 2: Principal component analysis score plot for the first two principal components of an analysis of 1H NMR spectra obtained from extracts of sunflower embryos from two genotypes (A and B) at different stages of development.
Figure 3: Principal component analysis loading plot identifying the contribution (weightings) of different spectral bins to the first principal component of 1H NMR spectra obtained from extracts of sunflower embryos at different stages of development.
Figure 4: Analysis of Ca2+ and Mg2+ levels in sunflower embryos at different stages of development.
Figure 5: Orthogonal PLS-DA (OPLS-DA) score plot of the first two principal components of an analysis of 1H NMR spectra obtained from extracts of embryos from different sunflower genotypes (A and B).
Figure 6: Orthogonal PLS-DA (OPLS-DA) weighting plot of the predictive component of an analysis of 1H NMR spectra obtained from extracts of embryos from different sunflower genotypes.

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Acknowledgements

This work was supported by the Biotechnology and Biological Sciences Research Council of the United Kingdom (grant # B17210), the Spanish 'Ministerio de Educación y Ciencia' and FEDER project AGL2005-00100 and funding from the Programa CSIC-I3P.

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Kruger, N., Troncoso-Ponce, M. & Ratcliffe, R. 1H NMR metabolite fingerprinting and metabolomic analysis of perchloric acid extracts from plant tissues. Nat Protoc 3, 1001–1012 (2008). https://doi.org/10.1038/nprot.2008.64

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