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, cultivars, development stages, pathogenic diseases, or environmental conditions (daily and seasonal changes, soils, water stress, mechanical stress, harvest and post- harvest treatments), among others, has been possible^3,4,5.

Mass spectrometry (MS) and Nuclear Magnetic Resonance (NMR) spectroscopy are the most widely used analytical platforms for this purpose. Contrary to MS methodologies, NMR stands as a highly reproducible, non-biased, quantitative, accurate, and non-destructive technique that requires minimal sample preparation, making it suitable for metabolomics studies. However, when compared to MS methods, the inherent low sensitivity is a limitation. In recent years and through the use of high-field magnets, cryogenic probes, micro-coil devices, and Dynamic Nuclear Polarization (DNP) methods, the sensitivity of NMR has been greatly improved. In the case of the latter approach, for instance, the sensitivity gain was in the level of two to three orders of magnitude^6,7. To date, almost 20% of the published metabolomics studies are NMR-based and the number is rising⁷.

Even though Proton NMR is the most popular and sensitive experiment for NMR metabolomics fingerprinting, it has some drawbacks. First, all the ¹H NMR signals detected in the sample are distributed in a small window corresponding to the proton chemical shift window, which results in crowded spectra. Second, the homonuclear scalar coupling splits the signals into multiple components (signal multiplicity), spreading the proton signal over a wider frequency range, complicating furthermore the spectra reading by increasing crowding and signal overlapping. In addition, NMR metabolomics is employed in the analysis of mixtures usually containing 50 to 300 molecules at an NMR observable concentration, generating complex spectra comprised of 200 to 2000 peaks.

Homonuclear decoupling proton NMR, also known as Pure Shift, is a method that induces the collapse of a multiplet signal into a single peak. It stands as an excellent tool for increasing signal resolution in crowded spectra^8,9,10 and therefore represents a convenient tool for plants metabolomics¹¹.

In the last decade, new Pure Shift pulse sequences, increasing both sensitivity and decoupling performance, have emerged. Their range of applications have also expanded, from molecular structure elucidation^12,13, to fluxomics¹⁴, mixture assignment^15,16,17, translational diffusion measurements¹⁸, enantiomeric discrimination¹⁹, unit distribution in co-polymers²⁰, among others.

Historically, Broadband Pure Shift experiments suffer from low sensitivity and complicated processing methods, limiting their scope in the assessment of biological extracts⁸. In 2014, Foroozandeh et al. published a new Pure Shift experiment, PSYCHE (Pure Shift Yielded by Chirp Excitation), based on anti-z-COSY pulse sequence which yielded excellent homonuclear decoupling and improved sensitivity values²¹. However, as PSYCHE is a 2D interferogram experiment where chunks of time domain data are acquired, it suffers from periodic sideband artifacts that result from J-coupling modulation distortions at the edges of the chunk. In complex mixtures, these artifacts yield signals larger than those associated with metabolites present at very low concentrations, hindering the analysis¹¹. There are two methods to remove these artifacts - TSE-PHYCHE²² and a more recent modification of the PSYCHE experiment called SAPPHIRE-PSYCHE (Sideband Averaging by Periodic PHase Incrementation of Residual J Evolution)²³.

In 2019, we demonstrated for the first time¹¹ that the SAPPHIRE-PSYCHE Pure Shift method, which removes artifacts with almost no sensitivity penalty²³, could be employed for the analysis of complex biological mixtures, such as extracts of the fruits of Physalis peruviana, commonly known as Cape gooseberries¹¹. We showed that these methods increase the performance of metabolomics data analyses such as metabolic assignment, correlation analysis and multivariate coefficients analysis¹¹. Since then, several Pure Shift metabolomics studies on different biological matrices, such as soft corals²⁴, hypericum plants²⁵, honey^26,27, tea²⁷, peppermint oil²⁶, and walnuts²⁸ have been addressed, demonstrating its importance as a new tool for metabolomics analysis. Paradoxically, the vast majority of these studies employed the standard and easy to implement PSYCHE pulse sequence, available from any spectrometer library, instead of the SAPPHIRE-PSYCHE pulse sequence, which has been shown to perform better. However, it requires better understanding of the pulse sequence for proper setup.

This paper is intended to help new users to apply Pure Shift methods in the study of plants, in particular, leaves of Vanilla sp (V. planifolia and V. pompona)²⁹, potato tubers (S. tuberosum)³⁰, and Cape gooseberries (P. peruviana)³¹. Sample preparation, NMR experimental set up, data acquisition, and data analysis are described in detail. Moreover, the protocol includes key notes to help researchers, new to the field, to properly set up PSYCHE and SAPPHIRE-PSYCHE experiments in the metabolomic profiling of plants.

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, and then centrifuge at 23,000 × g for 20 min at 10 °C.
   6. Recover the supernatant and filter it through a 13 mm polytetrafluoroethylene (PTFE) 0.45 µm syringe filter.
   7. Lyophilize 1 mL of the filtered extract to dryness and then re-suspend the obtained solid in 0.9 mL of 200 mM sodium oxalate buffer pH 4. Vortex.
   8. Lyophilize the resulting sample to dryness and dissolve it in 0.9 mL of deuterium oxide containing 5 mM of 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid sodium salt (TMSP-d₄).
   9. Fill the NMR tube with 0.6 mL of the sample using a micropipette.
2. Vanilla leaves
   1. Collect the leaves, clean them with damp tissue paper and freeze them whole in liquid nitrogen.
   2. Break the leaves into small pieces and lyophilize for 4 days until dryness.
   3. Grind the dry matter to a fine powder using an electric grinder.
   4. Weigh 50 mg of ground material and add 0.75 mL phosphate buffer pH 6.0 in deuterium oxide containing 0.1% of TMSP (w/w) and 0.75 mL methanol-d4. Vortex for 1 min.
   5. Sonicate for 20 min at 25 °C.
   6. Centrifuge at 13,000 × g for 10 min at 25 °C.
   7. Recover the supernatant (~1.3-1.4 mL) and filter it through a 13 mm PTFE 0.45 µm syringe filter.
   8. Fill the NMR tube with 0.6 mL of the filtered sample using a micropipette.
3. Potato tubers
   1. Peel and slice 4 to 8 tubers. Immediately, place approximately 125 g of material in stand-up bags and freeze them in liquid nitrogen.
      ​NOTE: To avoid oxidation during handling keep the potato damp.
   2. Lyophilize for 4 to 6 days until complete dryness.
   3. Grind the dry matter to a fine powder using an electric grinder.
   4. Weigh 160 mg of ground tuber and add 1.6 mL of deionized water. Vortex for 1 min.
   5. Sonicate for 45 min at 10 °C.
   6. Centrifuge at 23,000 x g for 20 min at 10 °C.
   7. Recover the supernatant (~1.5 -1.6 mL) and evaporate it to dryness in a refrigerated centrifugal vacuum concentrator for 16 h, at 10 °C.
   8. Add to the solid obtained (20-25 mg) 0.9 mL of 100 mM sodium oxalate buffer pH 4, vortex, and evaporate for 16 hours at 10 °C.
   9. Dissolve the solid obtained in 0.9 mL of deuterium oxide containing 3 mM of TMSP.
   10. Centrifuge at 23,000 x g for 5 min at 10 °C and filter the supernatant directly into the NMR tube through a 13 mm PTFE 0.45 µm syringe filter.
       ​NOTE: In this case, direct filtration into the NMR tube was performed to diminish steps in the preparation of more than 1000 samples.

2. NMR Data Acquisition and Processing

1. NMR initial setup
   1. Transfer the samples to the NMR spectrometer.
   2. Tune and match the probehead.
   3. Lock and shim the sample.
   4. Calibrate the 90° hard pulse. Calibrate the 90° pulse using any of the standard procedures.
   5. Run a standard 1D proton NMR spectrum.
2. PSYCHE experiment
   1. Select the reset_psyche_1d pulse sequence from the Bruker Topspin library (Figure S1). Use the following standard parameters: 5 kHz spectral width (SW2), at least 1 or 2 seconds of relaxation recovery delay (D1), 16 dummy scans (DS), 64 or 128 complex data points per block (L31), and 64 or 128 scans (NS) (Figure S1).
      NOTE: L31 is the number of complex digital points acquired in each Pure Shift block, best to be set to a power of 2.²¹
   2. Set the desired CHIRP pulse flip angle excitation (CNST61) and 10 kHz for the CHIRP pulse bandwidth (CNST60) (Figure S2).
      NOTE: The PSYCHE experiment is based on an anti-z-COSY scheme; consequently the CHIRP pulse flip angle needs to be small to avoid recoupling artifacts (Figure 1). The absolute intensity increases with the excitation flip angle. The periodic artifacts are also enhanced, spreading into the spectrum and increasing the "noise" (Figure 1). The "noise" becomes a combination of standard noise and chuncking artifacts. A good compromise between sensitivity and low recoupling artifacts is to set CNST61 = 20°.^19,22
   3. Set the hard pulse length (P1) to the previously calibrated value and the PSYCHE shape pulse length to 30 ms (P49) (Figure S2).
      NOTE: It is very important to calibrate the hard pulse value as the shape pulse powers will be automatically calculated from this value.
   4. Choose the Crp_psyche.20 (SPNAM 37) shape pulse for the PSYCHE element (Figure S2).
   5. Set the strength of the pulse field gradient applied during the PSYCHE element (GPZ0). Choose RECT.1 for the gradient shape pulse (GPNAM 0) (Figure S2).
      NOTE: A weak magnetic field gradient is applied during the PSYCHE element, normally, between 1% to 4% of the maximum strength of the gradient, depending on the probe.
   6. Set the number of blocks to acquire in order to reconstruct the Pure Shift FID (TD1) (Figure S3).
      NOTE: PSYCHE is acquired as a pseudo-2D experiment where TD1 is the number of Pure Shift interferogram blocks. The spectrum resolution depends on the size of the spectral window (SW1) and the total number of acquired points, which is TD1*2*L31. Typically, 16 or 32 blocks with 64 or 128 complex points per block will provide enough digital resolution. As PSYCHE is recorded in an interferogram manner, a higher number of blocks increase the digital resolution, but, also the total acquisition time¹⁹. Homonuclear J-couplings evolve during each block resulting in an oscillating J modulation pattern^21,23. After Fourier transform, this generates periodic sideband artifacts that depend on the length of the block (Figure 2). To reduce artifacts, the duration of the block must be short, typically less than 16 ms (block duration = 2*in0: Figure S1). If the block duration is high, reduce L31.
   7. Process the data with Bruker's Proc_reset AU program and Fourier transform.
      NOTE: We recommend to transform the spectrum using zero filling and a sine bell apodization (Figure S4).
3. SAPPHIRE-PSYCHE experiment
   1. Select the SAPPHIRE-PSYCHE pulse sequence and set the pulse sequence parameters. Standard parameters would be the following: 5 kHz spectral width (SW3), at least 1 or 2 seconds of relaxation delay (D1), 16 dummy scans (DS), 8 or 16 scans per increment (NS) and D2 to 14 ms (Figure S5).
      NOTE: This sequence is not in Bruker's repertoire, however, the sequence and the processing programs may be obtained from the Manchester NMR Methodology Group website, (https://www.nmr.chemistry.manchester.ac.uk/?q=node/426)²³. The D2 delay ensures that T2 relaxation remains constant with each J modulation increment. D2 needs to be greater than 1/4*SW1+p16+2*d16.²³
   2. Set the desired CHIRP pulse flip angle excitation (CNST20) and 10 kHz for the CHIRP pulse bandwidth (CNST21) (Figure S6).
      NOTE: As in the regular PSYCHE experiment, the CHIRP pulse flip angle needs to be short to avoid recoupling artifacts. CNST20 = 20° is a good compromise between sensitivity and low recoupling artifacts^21,23,25.
   3. Set the hard pulse length (P1) to the previously calibrated value and the PSYCHE shape pulse length to 30 ms (P40) (Figure S6).
      NOTE: It is important to calibrate the hard pulse value as the shape pulse powers will be automatically calculated from it.
   4. Choose the PSYCHE_Saltire_10kHz_30m shape pulse for the PSYCHE element (Figure S6).
   5. Set the strength of the pulse field gradient applied during the PSYCHE element (GPZ10). Choose RECT.1 for the gradient shape pulse (GPNAM 10) (Figure S7).
      NOTE: A weak magnetic field gradient is applied during the PSYCHE element, normally, between 1% and 4% of the maximum strength of the gradient, value that depends on the probe.
   6. Set the number of SAPPHIRE J modulation increments in F2 (TD2) (Figure S7).
      NOTE: normally 8 increments ensure an excellent suppression of sideband artifacts (Figure 2 and 3). The total number of scans of the final Pure Shift FID is NS*TD2.²³
   7. Set the F1 and F2 spectral windows (SW1 and SW2) (Figure S5).
      NOTE: SW2=SW3/(2*TD2) and SW3/SW1 = TD2*N, were TD2 and N are even integers²³. The SAPPHIRE-PSYCHE experiment is acquired as a pseudo 3D where F2 encodes the J-coupling artifact phase modulation and F1 the Pure Shift interferogram acquisition²⁰. Since SAPPHIRE-PSYCHE removes J modulation sidebands, the interferogram Pure Shift block duration could be longer than regular PSYCHE (Pure Shift block duration = 1/SW1), typically between 20 to 40 ms (Figure 2). However, longer chunk data acquisition leads to higher J-coupling evolutions, which would require more J-coupling phase modulation increments to remove the stronger sidebands attained²³.
   8. Set the number of Pure Shift blocks (TD1) (Figure S7).
      NOTE: Since SAPPHIRE-PSYCHE needs to compensate the J-coupling phase modulation of the first block, an extra block needs to be acquired. Typically, 17 (16+1) or 33 (32+1) blocks give enough digital resolution²³.
   9. Process the data executing the pm_pshift and the pm_fidadd AU programs followed by Fourier transform²³.
      NOTE: We recommend to transform the spectrum using zero filling and a sine bell apodization (Figure S4).

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