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

The aim of this procedure is to analyze plant extracts by pure shift NMR. The following protocol includes key aspects in the sample preparation from different plant matrices, vanilla leaves, potato tubers, and cape gooseberry fruits, and the detailed step-by-step NMR procedure to record optimal pure shift psyche and SAPPHIRE psyche spectra. Metabolic profiling by proton NMR is the art of analyzing signal by signal in the myriad of signals within the NMR spectra of a complex biological mixture with the aim of identifying each metabolite in this mixture.

The aim is to depict biomarkers that could be associated with chemotaxonomy, phenotyping, organoleptic properties, denomination of origin, metabolic responses among other important areas in plant sciences. Proton NMR is commonly used in metabolic profiling. However, the large number of signals with expand multiplicities confined in a narrow product spectral range leads to extensive overlap, complicating the analysis and the interpretation of the spectrum.

Typically, a single resonance has a width ranging from one to five Hertz. However, a highly coupled signal can spread in over 25 to even 50 Hertz, increasing the probability of signal overlap. To overcome this limitation, we apply modern pure shift NMR method to announce the spectral resolution as show here in three different plant scenarios.

Sample preparation, plant extract preparation can be performed in many ways and the procedure will depend on the matrix. The Cape gooseberry fruits were homogenized into a juice first. The vanilla leaves were used intact, and the potatoes were sliced protected from oxidation.

In all cases, the material was lyophilized, grinded and then extracted. For the cape gooseberry fruits and the potatoes water was used for the extraction aided by sonication. The supernatant was recovered by centrifugation, any liquid was dried by lyophilization or speed vac.

The dry extract was resuspended in oxalate buffer, evaporated to dryness and re dissolved in D2O containing TMSP. In the case of vanilla, the extraction was direct using deuterated phosphate buffer containing TMSP and deuterated methanol. The supernatant was also recovered by centrifugation.

All samples were filtered through a PTFE syringe filter, and the NMR tubes were filled with 0.6 milliliters of the filtered solution. Sample preparation in NMR metabolomics is key. Since the procedure will be repeated maybe hundreds of times, it should be prepared exactly in the same way, in order to guarantee that the variance observed is not due to sample preparation, but to real differences among the plants studied.

Pure shift NMR. The NMR spectrum is obtained after Fourier transform of the FID signal. The FID signal can be decomposed in two components, chemical shift modulation, and the J coupling modulation responsible for the J coupling splitting pattern.

The J coupling modulation could be refocused by a J coupling refocusing element, which selectively inverts passive spins while the active spins remain unaffected. After two equal delays the chemical J coupling is fully refocused. Psyche element based on an anti-z-COSY experiment is one of the most robust and sensitive broadband refocus elements, properties that render it suitable for NMR metabolomics.

Pure shift experiments are based on refocusing the J coupling evolution during chemical shift recording. This is typically accomplished by increasing the delays in order to move the J coupling refocusing point. As chemical shift evolves at a higher frequency than the J coupling, a homonuclear decoupled experiment can be recorded in an interferogram manner.

Interferogram acquisition, consists in recording the FID by small chunks with the refocusing point of the J coupling evolution, always coinciding with the center of the acquired chunk. The decoupled FID is constructed by concatenating each successive chunk. NMR data acquisition setup, transfer the samples to the NMR spectrometer.

Tune and match the probe head, lock and shim the sample. Calibrate the 90 degrees hard pulse. Run a standard 1D proton NMR spectrum.

Psyche experiment. Select the reset psyche 1D pulse sequence from the Bruker TopSpin library. Set the spectrum width to five kilohertz.

The relaxation recovery delay to at least one or two seconds. The dummy scans to 16. The number of scans to 64 or 128 and the number of complex data points per block, either 64 or 128, set the desired chirp pulse flip angle excitation.

A good between sensitivity and low recoupling artifacts is to set the constant 61 to 20 degrees, 10 kilohertz for the chirp pulse bandwidth. Set the hard pulse length to the previously calibrated volume and the psyche shape pulse length to 30 milliseconds. Choose the Crp_psyche.

20 shape pulse for the psyche element. The strength of the pulse field gradient applied during the psyche element is normally set between one to 4%of the maximum strength of the gradient depending on the probe. Choose RECT.

1 for the gradient shape pulse. Set the number of blocks to acquire to reconstruct the pure shift FID. Typically 16 or 32 blocks with 64 or 128 complex points per block will provide enough digital resolution, run the spectrum and process the data.

With Bruker's proc_reset AU program, and then Fourier transform. We recommend to transform the spectrum using zero filling and a sine bell apodization. Psyche is a pseudo 2D interferogram experiment.

It's software from periodic side span artifact arriving from the small J coupling evolution during the acquisition of each block that typically range from five to 20 milliseconds in the analysis of pure compound these artifacts can be neglected as they normally represent less than 5%of the parent peak. However, in complex mixtures, the side span artifact of some metabolite could be as large or larger than the signal of less concentrated metabolites compromising the accuracy of the metabolic analysis. These artifacts can be efficiently removed using a modification of psyche experiment called SAPPHIRE psyche developed in Morris Lab SAPPHIRE psyche experiment.

To attain the decoupled spectrum, the pulse sequence acquires small chunks of FID refocus in the J coupling at the middle of each block. However, a small J coupling evolution occurs during each block and generates the periodic side band artifacts. The SAPPHIRE psyche experiment is a modification of the regular psyche sequence in which these periodic artifacts are removed by systematic phase modulation, achieved by shift in the J refocusing point.

After adding each phase modulation, the residual J coupling evolution is highly suppressed, yielding a much cleaner pure shift spectrum. Select the SAPPHIRE psyche pulse sequence and set the pulse sequence parameters. 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.

The standard parameters are set to the following values, five kilohertz spectral width, at least one or two seconds of relaxation delay. 16 dummy scans, eight or 16 scans per increment, and D2 set to 14 milliseconds. This parameter ensures that the relaxation T2 remains constant with each J modulation increment.

Set the desired chirp pulse flip angle excitation value, and the pulse bandwidth. Set the hard pulse length to the previously calibrated value and the psyche shape pulse length to 30 milliseconds. Choose the PSYCHE_Saltire_10kHz_30m shape pulse for the psyche element.

Set the strength of the pulse field gradient applied during the psyche element. Choose RECT. 1 for the gradient shape.

Set the number of Sapphire J modulation increments in F2, normally eight increments ensure an excellent suppression of sideband artifacts. Set the F1 and F2 spectral windows that calculated from the selected F3 spectral window value using the following expressions. The pure shift block duration described as one over SW1 is typically set between 20 to 40 milliseconds.

Set the number of pure shift blocks. Since SAPPHIRE psyche needs to compensate the decoupling phase modulation of the first block an extra block needs to be added. Typically, 17 or 33 blocks give enough digital resolution.

Process the data, execute in the pm_pshift and the pm_fidadd AU programs followed by Fourier transform, we recommend to transform the spectrum using zero filling and assign bell apodization. Results, SAPPHIRE psyche experiments increase spectra resolution by collapsing coupled resonances into nice sharp singlets as seen demonstrated in three different plant matrices. Vanilla leaves, Physalis Peruviana fruits and potato tubers with SAPPHIRE Psyche complex multiplicities for example, the highly coupled hydrogens of homocitric acid that generate almost continuous signals expanding through 40 Hertz collapsed into three sharp singlets.

Reducing the crowding that could be masking other signals in the area. The gaining resolution also facilitated to clearly disentangle the highly overlapped region between 2.6 to 2.8 PPM in which homocitric acid is lactone and malic acid resonances appear. Key biomarkers in vanilla, for example, who signals overlap with those of glucosides in regular proton NMR were better identified in the SAPPHIRE spectra due to this outstanding gain in resolution.Conclusions.

Pure shift is an excellent new tool for plant metabolomics. It drastically increased the spectrum resolution and therefore allows an easier metabolite identification, a finer correlation metric analysis, and a better interpretation of multivariate analysis. After watching this video, you will have a good understanding of how to prepare different plan extract for NMR analysis and how to record optimal pure shift psyche and SAPPHIRE psyche spectrum.