This study was funded by the Consejo Nacional de Ciencia, Tecnolog&#237;a e Innovaci&#243;n Tecnol&#243;gica (CONCYTEC) - Programa Atracci&#243;n de Investigadores Cienciactiva - Contract # 008-2017-FONDECYT.

1. Sample preparation
<ol>
	<li>Cape gooseberries
	<ol>
		<li>Place 100-200 g of fresh&#160;fruits in a blender vase. Keep at 4 &#176;C for 30 min, and then homogenize in a laboratory blender.</li>
		<li>Immediately, transfer the juice to 50 mL plastic tubes, freeze them in liquid nitrogen and lyophilize to dryness for 4 to 5 days.</li>
		<li>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.</li>
		<li>Weigh 1 g of the ground material and add 10 mL of ultrapure water. Vortex for 1 min.</li>
		<li>Sonicate for 20 min at 10 &#176;C, and then centrifuge at 23,000 &#215; g for 20 min at 10 &#176;C.</li>
		<li>Recover the supernatant and filter it through a 13 mm polytetrafluoroethylene (PTFE) 0.45 &#181;m syringe filter.</li>
		<li>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.</li>
		<li>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).</li>
		<li>Fill the NMR tube with 0.6 mL of the sample using a micropipette.</li>
	</ol>
	</li>
	<li>Vanilla leaves
	<ol>
		<li>Collect the leaves, clean them with damp tissue paper and freeze them whole in liquid nitrogen.</li>
		<li>Break the leaves into small pieces and lyophilize for 4 days until dryness.</li>
		<li>Grind the dry matter to a fine powder using an electric grinder.</li>
		<li>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.</li>
		<li>Sonicate for 20 min at 25 &#176;C.</li>
		<li>Centrifuge at 13,000 &#215; g for 10 min at 25 &#176;C.</li>
		<li>Recover the supernatant (~1.3-1.4 mL) and filter it through a 13 mm PTFE 0.45 &#181;m syringe filter.</li>
		<li>Fill the NMR tube with 0.6 mL of the filtered sample using a micropipette.</li>
	</ol>
	</li>
	<li>Potato tubers
	<ol>
		<li>Peel and slice 4 to 8 tubers. Immediately, place approximately 125 g of material in stand-up bags and freeze them in liquid nitrogen. 
		&#8203;NOTE: To avoid oxidation during handling keep the potato damp.</li>
		<li>Lyophilize for 4 to 6 days until complete dryness.</li>
		<li>Grind the dry matter to a fine powder using an electric grinder.</li>
		<li>Weigh 160 mg of ground tuber and add 1.6 mL of deionized water. Vortex for 1 min.</li>
		<li>Sonicate for 45 min at 10 &#176;C.</li>
		<li>Centrifuge at 23,000 x g for 20 min at 10 &#176;C.</li>
		<li>Recover the supernatant (~1.5 -1.6 mL) and evaporate it to dryness in a refrigerated centrifugal vacuum concentrator for 16 h, at 10 &#176;C.</li>
		<li>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 &#176;C.</li>
		<li>Dissolve the solid obtained in 0.9 mL of deuterium oxide containing 3 mM of TMSP.</li>
		<li>Centrifuge at 23,000 x g for 5 min at 10 &#176;C and filter the supernatant directly into the NMR tube through a 13 mm PTFE 0.45 &#181;m syringe filter. 
		&#8203;NOTE: In this case, direct filtration into the NMR tube was performed to diminish steps in the preparation of more than 1000 samples.</li>
	</ol>
	</li>
</ol>
2. NMR Data Acquisition and Processing
<ol>
	<li>NMR initial setup
	<ol>
		<li>Transfer the samples to the NMR spectrometer.</li>
		<li>Tune and match the probehead.</li>
		<li>Lock and shim the sample.</li>
		<li>Calibrate the 90&#176; hard pulse. Calibrate the 90&#176; pulse using any of the standard procedures.</li>
		<li>Run a standard 1D proton NMR spectrum.</li>
	</ol>
	</li>
	<li>PSYCHE experiment
	<ol>
		<li>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</li>
		<li>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 &#34;noise&#34; (Figure 1). The &#34;noise&#34; becomes a combination of standard noise and chuncking artifacts. A good compromise between sensitivity and low recoupling artifacts is to set CNST61 = 20&#176;.19,22</li>
		<li>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.</li>
		<li>Choose the Crp_psyche.20 (SPNAM 37) shape pulse for the PSYCHE element (Figure S2).</li>
		<li>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.</li>
		<li>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.&#160;Homonuclear J-couplings evolve during each block resulting in an oscillating J modulation pattern21,23. 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.</li>
		<li>Process the data with Bruker&#39;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).</li>
	</ol>
	</li>
	<li>SAPPHIRE-PSYCHE experiment
	<ol>
		<li>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&#39;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.&#160;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</li>
		<li>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&#176; is a good compromise between sensitivity and low recoupling artifacts21,23,25.</li>
		<li>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.</li>
		<li>Choose the PSYCHE_Saltire_10kHz_30m shape pulse for the PSYCHE element (Figure S6).</li>
		<li>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.</li>
		<li>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</li>
		<li>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.</li>
		<li>Set the number of Pure Shift blocks (TD1) (Figure S7). 
		NOTE: Since SAPPHIRE-PSYCHE needs to compensate the J-coupling&#160;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.</li>
		<li>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).</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to declare.

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...

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 environment1,2. 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 possible3,4,5.
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 magnitude6,7. 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 spectra8,9,10 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 elucidation12,13, to fluxomics14, mixture assignment15,16,17, 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, honey26,27, 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.

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

pure shift nuclear magnetic resonance: a new tool for plant metabolomics

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&#160;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&#160;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.

NMR spectrum analysis 
PSYCHE experiments increase spectra resolution by collapsing coupled resonances into singlets21,&#160;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 Figur...

Watch this Scientific Journal Video about Pure Shift Nuclear Magnetic Resonance: a New Tool for Plant Metabolomics at JoVE.com

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

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.

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 ...

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Research

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Biochemistry

1.7K Views.  Pontificia Universidad Católica del Perú. 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.

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

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Immunology and Infection

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Environment

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which are involved in many cell activities. Here, we describe a rapid and reliable method for the absolute qualification of nucleoside/nucleotide contents in plants. Briefly, 100 mg of homogenized plant material was extracted with 1 mL of extraction buffer (methanol, acetonitrile, and water at a ratio of 2:2:1). Later, the sample was concentrated five times in a freeze dryer and then injected into an HPLC-MS/MS. Nucleotides were separated on a porous graphitic carbon (PGC) column and nucleosides were separated on a C18 column. The mass transitions of each nucleoside and nucleotide were monitored by mass spectrometry. The contents of the nucleosides and nucleotides were quantified against their external standards (ESTDs). Using this method, therefore, researchers can easily quantify nucleosides/nucleotides in different plants.

A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Neuroscience

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Biology

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Metabolomics Using NMR Spectroscopy to Identify Dysregulated Metabolites

Identification and Quantification of Deranged Metabolites in Critically Ill Patients Using NMR-Based Metabolomics

Metabolomics is emerging as a significant approach to reflect the individual's response to pathophysiological conditions. Nuclear magnetic resonance (NMR) spectroscopy has evolved as a tool to identify metabolic dysregulations in critically ill patients afflicted with conditions like acute respiratory distress syndrome (ARDS), severe acute pancreatitis (SAP), acute kidney injury (AKI), and sepsis. The spectral data from the serum sample of the study and control group are recorded using an 800 MHz NMR spectrometer and processed using NMR processing and analysis tools. Furthermore, a rigorous statistical analysis, such as univariate and multivariate tests, is performed to pinpoint significant metabolites, which are then accurately identified and quantified using NMR metabolite quantification software. Additionally, pathway analysis highlights the deranged biochemical cycles that result in the severity of illness. Through this comprehensive approach, researchers aim to gain deeper insights into the metabolic alterations associated with these critical illnesses, potentially paving the way for a better understanding of the disease and improved diagnostics and treatment strategies.

Nuclear magnetic resonance (NMR) spectroscopy is used to identify dysregulation in the metabolites in patients with various diseases. This technique allows the quantification of the deranged metabolites, unraveling the pathophysiological insights. Here, we describe the step-by-step procedure of the NMR-based approach for the metabolic characterization of the patients.

Metabolomics is emerging as a significant approach to reflect the individual's response to pathophysiological conditions. Nuclear magnetic resonance (NMR) ...

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

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Chapters in this video

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by ¹H NMR Spectroscopy

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Identification and Quantification of Deranged Metabolites in Critically Ill Patients Using NMR-Based Metabolomics