Analysis of plant secondary metabolism using stable isotope‐labelled precursors

Introduction Analysis of biochemical pathways typically involves feeding a labelled precursor to an organism, and then monitoring the metabolic fate of the label. Initial studies used radioisotopes as a label and then monitored radioactivity in the metabolic products. As analytical equipment improve...

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Bibliographic Details
Published in:Phytochemical analysis 2021-01, Vol.32 (1), p.62-68
Main Authors: Arroo, Randolph R.J., Bhambra, Avninder S., Hano, Christophe, Renda, Gülin, Ruparelia, Ketan C., Wang, Meng F.
Format: Article
Language:English
Subjects:
Atomic properties
biosynthesis
biosynthetic pathways
Carbon 13
Carbon Isotopes
Chromatography
Chromatography, Liquid
Deuterium
Feeding
Gas chromatography
Gas Chromatography-Mass Spectrometry
Isotopes
Labels
Life Sciences
Liquid chromatography
Magnetic Resonance Spectroscopy
Mass spectrometry
Mass spectroscopy
Metabolic flux
Metabolism
Metabolites
Metabolomics
NMR
Nuclear magnetic resonance
Precursors
Radioactivity
Radioisotopes
Secondary Metabolism
Stable isotopes
Tandem Mass Spectrometry
terpenoids
Toolkits
tropane alkaloids
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Summary:Introduction Analysis of biochemical pathways typically involves feeding a labelled precursor to an organism, and then monitoring the metabolic fate of the label. Initial studies used radioisotopes as a label and then monitored radioactivity in the metabolic products. As analytical equipment improved and became more widely available, preference shifted the use stable ‘heavy’ isotopes like deuterium (2H)‐, carbon‐13 (13C)‐ and nitrogen‐15 (15N)‐atoms as labels. Incorporation of the labels could be monitored by mass spectrometry (MS), as part of a hyphenated tool kits, e.g. Liquid chromatography (LC)–MS, gas chromatography (GC)–MS, LC–MS/MS. MS offers great sensitivity but the exact location of an isotope label in a given metabolite cannot always be unambiguously established. Nuclear magnetic resonance (NMR) can also be used to pick up signals of stable isotopes, and can give information on the precise location of incorporated label in the metabolites. However, the detection limit for NMR is quite a bit higher than that for MS. Objectives A number of experiments involving feeding stable isotope‐labelled precursors followed by NMR analysis of the metabolites is presented. The aim is to highlight the use of NMR analysis in identifying the precise fate of isotope labels after precursor feeding experiments. As more powerful NMR equipment becomes available, applications as described in this review may become more commonplace in pathway analysis. Conclusion and Prospects NMR is a widely accepted tool for chemical structure elucidation and is now increasingly used in metabolomic studies. In addition, NMR, combined with stable isotope feeding, should be considered as a tool for metabolic flux analyses. Heavy isotopes, like deuterium (2H)‐, carbon‐13 (13C)‐ and nitrogen‐15 (15N)‐atoms, are regularly used as labels in precursor feeding experiments. Mass spectrometry is widely used to monitor the metabolic fate of the labelled precursors. However, this analytical tool does not always allow to pinpoint the exact location of the isotope label in any given metabolite. Nuclear magnetic resonance (NMR) also picks up signals of stable isotopes, but in addition provides information on the precise location of incorporated label in the metabolites. This makes the latter an attractive tool for biosynthetic studies and metabolic flux analysis.
ISSN:0958-0344
1099-1565
DOI:10.1002/pca.2955