Pure Shift Nuclear Magnetic Resonance: a New Tool for Plant Metabolomics

Summary

This paper presents the use of PSYCHE and SAPPHIRE-PSYCHE in the metabolic profiling of plants and includes detailed procedures for sample preparation and optimal Pure Shift NMR spectra recording. Examples through which the gain in resolution achieved by homonuclear decoupling allows a more comprehensive understanding of the system are discussed.

Abstract

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not only provides quantitative data but also permits structural identification of the metabolites present in complex mixtures.

Metabolic profiling by ¹H NMR has proven useful in the study of various types of plant scenarios, which include the evaluation of crop conditions, harvest and post- harvest treatments, metabolic phenotyping, metabolic pathways, gene regulation, identification of biomarkers, chemotaxonomy, quality control, denomination of origin, among others. However, signal overlapping of the large number of resonances with expanded J-coupling multiplicities complicates the spectra analysis and its interpretation, and represents a limitation for classical ¹H NMR profiling.

In the last decade, novel NMR broadband homonuclear decoupling techniques through which multiplet signals collapse into single resonance lines - commonly called Pure Shift methods - have been developed to overcome the spectra resolution problem inherent to ¹H NMR classical spectra.

Here a step-by-step protocol of the plant extract preparation and the procedure to record optimal Pure Shift PSYCHE and SAPPHIRE-PSYCHE spectra in three different plant matrices - Vanilla plant leaves, potato tubers (S. tuberosum), and Cape gooseberries (P. peruviana) - is presented. The effect of the gain in resolution in metabolic identification, correlation analysis and multivariate analyses, as compared against classical spectra, is discussed.

Introduction

The complete set of metabolites that comprise an organism - substrates, intermediates, and end products of biological processes - was coined in 1998 with the term, metabolome. It is well known that the metabolome is closely related to the phenotype, and it is of particular interest in plants as it reflects the direct interaction between the genotype and the environment^1,2. Hence, the characterization of the metabolomic profile has become of paramount importance in plants. Through the identification and quantification of biomarkers (key metabolites) and metabolic patterns, the discrimination between species, cu....

Protocol

1. Sample preparation

1. Cape gooseberries
   1. Place 100-200 g of fresh fruits in a blender vase. Keep at 4 °C for 30 min, and then homogenize in a laboratory blender.
   2. Immediately, transfer the juice to 50 mL plastic tubes, freeze them in liquid nitrogen and lyophilize to dryness for 4 to 5 days.
   3. Grind the lyophilized material to a fine powder using an electric grinder.
      NOTE: Handling of the dry material needs to be done quickly because the powder is highly hygroscopic.
   4. Weigh 1 g of the ground material and add 10 mL of ultrapure water. Vortex for 1 min.
   5. Sonicate for 20 min at 10 °C, an....

Results

NMR spectrum analysis
PSYCHE experiments increase spectra resolution by collapsing coupled resonances into singlets²¹, which in turn reduces overlap and facilitates assignment and data analysis. Pure Shift NMR can be applied to plant extracts. Here we demonstrate its use in three different matrices: vanilla leaves, potato tubers, and Physalis peruviana fruits. The resolution enhancement achieved in the spectra of these plant extracts is clear from Figures S8-S.......

Discussion

Metabolite structural identification and quantitation are key issues in the characterization of the metabolome, data that when subjected to multivariable analyses permits to better understand the biological system under study. Sample preparation and data acquisition are critical aspects that need optimization in order to provide reliable results.

In this article, we describe and illustrate the sample preparation for NMR analysis of three different plant matrices. As with any extraction procedu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
77500 Series Freezone 4.5 Liter benchtop	Labconco	77500
Bruker Avance III 500 MHz equiped with a 5 mm TCI Z-gradient cryogenic probe	Bruker Corporation
Centrivap Refrigerated Centrifugal Concentrators Labconco 7310000 Series	Labconco	7310000
Deuterium oxide	Sigma-Aldrich	151882
Grinder machine MKM6003	Bosch	MKM6003
Licuadora Blender 8011S model Hgb2wts3	Waring	Hgb2wts3
Methanol-d4	Sigma-Aldrich	151947