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.

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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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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

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

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 ...

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known small molecule-protein interactions and to find potential protein targets for natural products. Compared with other methods, DARTS uses native, unmodified, small molecules and is simple and easy to operate. In this study, we further enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The protein-ligand interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used the mTOR-rapamycin interaction as an exemplary case for establishment of our protocol. From the proteolytic curve we saw that the proteolysis of mTOR by pronase was inhibited by the presence of rapamycin. The dose-dependency curve allowed us to estimate the binding affinity of rapamycin and mTOR. This method is likely to be a powerful and simple method for accurately identifying novel target proteins and for the optimization of drug target engagement.

A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction

In this study, we enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used mTOR-rapamycin interaction as an exemplary case.

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...