# Top PDF High-resolution Magic-angle Spinning (HR-MAS) NMR Spectroscopy

### High-resolution Magic-angle Spinning (HR-MAS) NMR Spectroscopy

where m is the sample mass, ω r is the spinning frequency, and r is the radial distance between the rotation axis and the point. As shown in the equation, F c increases with both the spinning frequency and sample diameter. Besides the centrifugal force, sample heating is also a potential draw- back of fast MAS as the friction-induced heat reduces the spectral resolu- tion and alters the metabolic activities. Slow MAS reduces the effect of both the centrifugal force and sample heating; however, special pulse sequences are often necessary to suppress the spinning sidebands. Moreover, at slow MAS rates the overall signal intensity is distributed throughout the spin- ning sidebands, reducing the sensitivity of metabolite detection. Various works have demonstrated that applying the 2D pASS sideband suppression pulse sequence, 41 it is possible to acquire MAS spectra of living bacterial cells 50 and animal tissues 51,52 at a spinning speed of only 40 Hz. For even lower spinning ( i.e., down to 1 Hz), a more sophisticated pulse experiment

### Evaluation de deux nouveaux outils pour l'étude d'échantillons biologiques par spectroscopie de Résonance Magnétique Nucléaire: -La RMN HR-MAS (High-Resolution Magic Angle Spinning): une technique d'analyse d'échantillons bruts ; -La Métabonomique: une méthode d'analyse statistique des spectres pour la recherche de signatures métaboliques

samples; -Metabonomic: a new statistical method of spectra analysis for research of metabolic signatures. The improvement of analytical tools is essential to find new metabolic biomarkers, to help for pathology diagnosis or to follow therapeutic effects. An extraction procedure is needed to analyse tissues by liquid high-resolution NMR. With HR-MAS (High-Resolution Magic Angle Spinning) NMR, a direct analysis of tissues is possible (animal or vegetal tissues). The first part of this work shows the different studies we carried out to evaluate the interests and the limitations of this new method. A critical evaluation, achieved on a large scale of biological samples (live animals (earthworm, ground bettle larvae), fresh and frozen kidney, cardiac and musculary tissues) highlights several drawbacks on logistical, sanitary and analytical points of view. HR-MAS NMR allows the simultaneous observation of hydrosoluble and organosoluble metabolites but their quantification is often difficult. Then rapid rotation of the rotor (1-5 kHz) involves sample alteration. The main advantage of this method resids in its ability to analyse small samples (12-92 µL) with high sensitivity. Our study led us to favor liquid NMR analysis on extract tissues rather than NMR analysis on intact tisssues. The second part concerns metabonomic by NMR. A compararive metabonomic study of human glioblastoma cell lines, radioresistant or radiosensitive, reveals a characteristic biomarker of radioresistance. Normalized principal component analysis (PCA) has been used for a statistical analysis of data obtained on different kinds of samples or with different analytical methods. Metabolic modifications induced by obesity on rat have been characterized by the combination of 1 H NMR spectra of hydrosoluble and organosoluble fractions. Characterization of phenotypes from different varieties of squash has been conforted by combination of data obtained with 1 H and 31 P NMR spectra.

### Intact NMR spectroscopy: slow high-resolution magic angle spinning chemical shift imaging

by 1 H high resolution-magic angle spinning (HR-MAS) NMR, Food Chemistry 2017, 233, 391– 400; [garlic] (a) Tingfu Liang, Feifei Wei, Yi Lu, Yoshinori Kodani, Mitsuhiko Nakada, Takuya Miyakawa, and Masaru Tanokura, Comprehensive NMR Analysis of Compositional Changes of Black Garlic during Thermal Processing, J. Agric. Food Chem. 2015, 63, 683−691; (b) Covadonga Lucas-Torres, Gaspard Huber, Atsuyuki Ichikawa, Yusuke Nishiyama, Alan Wong, HR-μMAS NMR-Based Metabolomics: Localized Metabolic Profiling of a Garlic Clove with μg Tissues, Anal. Chem. 2018, 90, 13736−13743. [zucchini] Ana Cristina Abreu, Luis Manuel Aguilera-Saéz, Araceli Peña, Mar García-Valverde, Patricia Marín, Diego L. Valera, Ignacio Fernańdez, NMR-Based Metabolomics Approach To Study the Influence of Different Conditions of Water Irrigation and Greenhouse Ventilation on Zucchini Crops, J.

### 1H high resolution magic-angle coil spinning (HR-MACS) μNMR metabolic profiling of whole Saccharomyces cervisiae cells: a demonstrative study

NMR is an inherently insensitive technique, thus HR-MAS analysis often relies on large sample volumes for detection; typ- ically about 100 million whole cells in a 30–50 μl volume for each of 3–5 replicate samples (for statistical analyses). In cases where sample size is limited (such as neuron cells), analysis of fewer cells—in a smaller volume—would ease the sample prepa- ration and may improve the high-throughput efficiency (e.g., coupling with micro-fluidic devices for cell separation tech- niques). One promising approach for volume-, or mass-, limited bio-specimens is the uses of a high-resolution magic-angle coil spinning (HR-MACS) ( Wong et al., 2012, 2013 ). HR-MACS, as with the original MACS experiment ( Sakellariou et al., 2007 ), utilizes a secondary tuned circuit and a simple and robust rotor insert, designed to fit inside a standard MAS sample rotor to convert the MAS probe into a μMAS probe without any probe modification. HR-MACS can be readily coupled with any stan- dard HR-MAS probe making it assessable to any laboratory with HR-MAS facilities. The use of a filling-factor optimized detector, susceptibility-optimized inserts and simultaneous spinning of the sample and detector have been demonstrated to yield high sensi- tivity and excellent spectral resolution (up to 2 ppb) allowing for high-precision metabolomic assessments of a sample volume less than 500 nl ( Wong et al., 2013 ). Moreover, the reduced diameter of the sample also abates the centrifugal forces exerted on the cells under sample spinning diminishing the chances of cell lysis due to sample spinning.

### Two- and Three-Dimensional Multinuclear Stray-Field Imaging of Rotating Samples with Magic-Angle Spinning (STRAFI-MAS): From Bio to Inorganic Materials

frequency and t rp is the rotor projection period used in the experiment), 0  and 90  , do correspond mod- estly with the view projection of the bone. For exam- ple, the image with a 90  rotor projection corresponds to a mirror reflection of the photograph; whereas, the image with 0  rotor projection corresponds to a 90  turn of the photographic picture. The visible distor- tions in the images arise from the fact that the experi- ments were carried out in an unstable MAS rotation, 1050 Hz with a constant fluctuation of 6 5 Hz. A bet- ter image quality could be obtained with a slower and a more stable MAS rotation. This could be achieved by using a larger MAS rotor with a turbine-less cap. It is interesting to note that Tritt-Goc et al (16) have acquired high spatial 3D resolution images (60  60  60 mm) of a human trabecular bone at 7T to investi- gate a bone disease called osteoporosis. In the study, a field gradient of 1 T/m was used along the x, y, and z direction, which is comparable to what we applied here for the tibia bone.

### Spatially-resolved metabolic profiling of living Drosophila in neurodegenerative conditions using 1H magic angle spinning NMR

between Gal4 drivers or UAS effectors with w 1118 . High-resolution MAS 1 H NMR spectroscopic imaging. The 1 H HRMAS NMR experiments on living Drosophila were performed essentially as previously described 8 , except that two flies were studied simultaneously. Two females were placed carefully in two separate compartments of a proton-free (PCTFE) cylindrical insert (top and bottom sides having a dedicated entrance) matching the size of the flies (~1 mm). The insert was then intro- duced in the center of the zirconia rotor so that the anteroposterior direction of the insects is aligned along the spinning axis. Anesthesia was obtained by maintaining the flies at 4 °C. The hardware and experimental parame- ters were identical to those described in Sarou-Kanian et al. 8 the 1 H HRMAS NMR spectra were recorded using a

### Solid-state 31P and 1H chemical MR micro-imaging of hard tissues and biomaterials with magic angle spinning at very high magnetic field

of rigid tissues and related biomaterials at very high magnetic field, with greatly improved signal to noise ratio and spatial resolution when compared to static conditions. Cross-polarization is employed to enhance contrast and to further depict spatially localized chemical variations in reduced experimental time. In these materials, very high magnetic field and moderate MAS spinning rate directly provide high spectral resolution and enable the use of frequency selective excitation schemes for chemically selective imaging. These new possibilities are exemplified with experiments probing selectively the 3D spatial distribution of apatitic hydroxyl protons inside a mouse tooth with attached jaw bone with a nominal isotropic resolution nearing 100 µm.

### Studying intact bacterial peptidoglycan by proton-detected NMR spectroscopy at 100 kHz MAS frequency

Newcastle University, Richardson Road, Newcastle upon Tyne, NE2 4AX, United Kingdom Abstract The bacterial cell wall is composed of the peptidoglycan (PG), a large polymer that maintains the integrity of the bacterial cell. Due to its multi-gigadalton size, heterogeniety, and dynamics, atomic-resolution studies are inherently complex. Solid-state NMR is an important technique to gain insight into its structure, dynamics and interactions. Here, we explore the possibilities to study the PG with ultra-fast (100 kHz) magic-angle spinning NMR. We demonstrate that highly resolved spectra can be obtained, and show strategies to obtain site- specific resonance assignments and distance information. We also explore the use of proton-proton correlation experiments, thus opening the way for NMR studies of intact cell walls without the need for isotope labeling.

### Assessment of magic angle spinning spectroscopy for studying migration in solid milk chocolate

All MAS experiments were conducted using a hr-MAS Bruker probe in the same 300 MHz system as the relaxation experiments. After loading the MAS sample rotor, the sample was[r]

### Accurate Determination of Interstrand Distances and Alignment in Amyloid Fibrils by Magic Angle Spinning NMR

determination of a high resolution structure to be achieved rather than inferring a model from a limited number of structural restraints. We have also compared our results to those reported previously for Aβ peptides 16 , 17 , which reveal that our distances are significantly shorter (~0.3-1.4 Å) than these cited values. Furthermore, the method may yield systematically smaller values of distances when compared to X-ray crystallographic measurements, as indicated by the succinate distance which is ~4% shorter than the distance obtained by X-ray diffraction measurements (3.76 Å vs. 3.91) 21 . If one assumes that internuclear distances in the fibrils systematically diverge from those measured by X-ray crystallography due to intrinsic differences in the methods, then the scaling by 4% results in our measurement of the average inter-fibril distance of 4.50 ± 0.11 scaling to 4.68 ± 0.11 agrees within the experimental error with those measured by powder diffraction (4.7 Å) 58 . Powder diffraction, however, lacks site-specific resolution and therefore cannot distinguish between the many possible topological arrangements of strands in the fibrils. Finally, it is also interesting to compare our data qualitatively to those reported by Balbach et al. using the constant-time RFDR sequence. On qualitative grounds, the agreement between data and experiment is significantly better for DQF-DRAWS, particularly at longer mixing times, and this is reflected in the accuracy of distances extracted by both methods. This may be due in part to the reduced sensitivity of DQF- DRAWS to relaxation as compared to RFDR, together with our fitting method that treats both the dipolar coupling and relaxation in a single global fit.

### Mobility of organic pollutants in soil components. What role can magic angle spinning NMR play ?

The FTIR spectra of pure and exchanged montmorillonites were similar (data not shown). The content of physisorbed water was greater after adsorption of PMG. Vibrational bands of PMG were systematically masked by those of the clays. Consequently, FTIR spectroscopy is not an appropriate method in this case. Thus, we recorded 1 H MAS Hahn echo spectra with a 1 ms echo time (data not shown). However, mobile water resonance dominated the spectra, avoiding unambiguous data analysis. The same samples were subsequently hydrated (200% w/w) and analysed by 1 H 1D HR-MAS NMR. The primary aim was to mimic environmental conditions more reasonably, in which water content usually plays an important role in the fate of pollutants. Moreover, it thus may be possible to increase the mobility of the adsorbed PMG. By this means, we expected sharper signals. The 1 H HR-MAS spectra of hydrated samples are presented in Figure 3(b–d). Resonances in the range 2.1–2.3 p.p.m. belong to 'impurities' of montmorillonite 'salted out' during adsorption of PMG. These resonances were not present in the PMG solution and they were not observed when montmorillonite was analysed just after swelling (Figure 3a). The major features of these spectra were as follows. (i) The high resolution of the spectra containing PMG, with its resonances clearly observed at 3.24 and 3.82 p.p.m., the coupling constant (11.5 Hz) being observed on some spectra. (ii) PMG signal intensities increased with the amount of PMG loaded onto the surface. It indicates that NMR preserves its quantitative properties despite the heterogeneous state of the samples. (iii) Signals were rather sharp, suggesting a relatively weak interaction between PMG and the negatively charged surface. This hypothesis is reasonable bearing in mind that PMG must interact with the montmorillonite surface by its ammonium moiety, contrary to previously published studies on goethite in which PMG entered the coordination sphere of iron via one of its phosphonate oxygens (Sheals et al., 2002). Further experiments are in progress in our group to confirm the nature of the interaction of PMG with the surface of this montmorillonite.

### Spatially-resolved metabolic profiling of living Drosophila in neurodegenerative conditions using 1H magic angle spinning NMR

between Gal4 drivers or UAS effectors with w 1118 . High-resolution MAS 1 H NMR spectroscopic imaging. The 1 H HRMAS NMR experiments on living Drosophila were performed essentially as previously described 8 , except that two flies were studied simultaneously. Two females were placed carefully in two separate compartments of a proton-free (PCTFE) cylindrical insert (top and bottom sides having a dedicated entrance) matching the size of the flies (~1 mm). The insert was then intro- duced in the center of the zirconia rotor so that the anteroposterior direction of the insects is aligned along the spinning axis. Anesthesia was obtained by maintaining the flies at 4 °C. The hardware and experimental parame- ters were identical to those described in Sarou-Kanian et al. 8 the 1 H HRMAS NMR spectra were recorded using a

### De novo prediction of cross-effect efficiency for magic angle spinning dynamic nuclear polarization

1. Introduction Dynamic nuclear polarization (DNP) attracts large interest because it can provide impressive sensitivity gains to solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy. In DNP, the large intrinsic polarization of electron spins is transferred to nuclei. While this method was established under static conditions, its extension to spinning samples 1–3 and high magnetic field (> 5 T) combined with high-power, high-frequency microwaves 4,5 has provided large nuclear hyperpolarization along with high spectral resolution for ssNMR. This has enabled NMR experiments that would have been otherwise impracticable 6–14 . It was with the introduction of nitroxide biradicals 15 that the highest Magic Angle Spinning (MAS) NMR sensitivities have been obtained using DNP. Based on the cross-effect (CE) mechanism under MAS, which requires two coupled electron spins and a mutually-coupled nuclear spin, 16–18 these bis-nitroxides can provide a fast build-up of relatively high nuclear polarization levels. Nowadays, two main categories of bis-nitroxides are being developed, based on different chemical linkers that tether the two nitroxide moieties: the bTbK family (based on a bis-Ketal linker), 19 and the bTurea family (based on a urea linker). 20,21 The chemical structures for these families were designed based on empirical approaches that have shown that both the relative orientation of the g-tensors of the two electron spins and their inter-spin distance affects the DNP efficiency. 19,20,22,23 Each family now has several substructures and colossal synthesis and experimental work has been performed to optimize the structures further. 23–27 These studies notably highlighted that electron relaxation times are key parameters for efficient DNP, 22 which can be tuned by changing the molecular weight of the radical. 28 Nowadays, derivatives of the bTbK 25,29 and bTurea 26,27,30 families, in particular TEKPol 24 and AMUPol, 23 are commonly used and considered as highly- efficient and versatile polarizing agents (PAs) for CE-DNP.

### Dynamic Nuclear Polarization Efficiency Increased by Very Fast Magic Angle Spinning

ABSTRACT: Dynamic nuclear polarization (DNP) has recently emerged as a tool to enhance the sensitivity of solid-state NMR experiments. However, so far high enhancements (>100) are limited to relatively low magnetic ﬁelds, and DNP at ﬁelds higher than 9.4 T signi ﬁcantly drops in eﬃciency. Here we report solid-state Overhauser e ﬀect DNP enhancements of over 100 at 18.8 T. This is achieved through the unexpected discovery that enhancements increase rapidly with increasing magic angle spinning (MAS) rates. The measurements are made using 1,3-bisdiphenylene-2-phenylallyl dissolved in o-terphenyl at 40 kHz MAS. We introduce a source −sink diﬀusion model for polarization transfer which is capable of explaining the experimental observations. The advantage of this approach is demonstrated on mesoporous alumina with the acquisition of well-resolved DNP surface- enhanced 27 Al cross-polarization spectra.

### Determination of long-range distances by fast magic-angle-spinning radiofrequency-driven 19 F-19 F dipolar recoupling NMR

To investigate how δ-pulse RFDR and fpRFDR differ in their distance resolution and sensitivity to the 19 F CSA tensor orientation, we simulated the buildup curves of a two-spin system, using the F O and F M spins in sitagliptin as a proxy but varying the F O CSA tensor orientation angle β from 0° to 90° (Fig. 8). The buildup curves were simulated for internuclear distances of 5.4 Å to 9 Å under different MAS frequencies and 19 F rf field strengths. Fig. 8a shows buildup curves for 15 kHz MAS and 83 kHz 19 F rf fields (f = 9%), representing the δ-pulse RFDR limit. It can be seen that the buildup curves are easily distinguishable with better than 0.5 Å resolution, and the distinction is much larger than the spread caused by the CSA tensor orientation, consistent with the experimentally measured distance accuracy for 14.9 kHz MAS (Fig. 5). However, the low spectral sensitivity at 14.9 kHz MAS due to CSA sidebands and fast relaxation makes this condition untenable for distance measurements. At 25 and 38 kHz MAS for pulse fractions of 15% and 30%, simulations indicate larger sensitivity to the 19 F CSA, with a distance resolution of 1 Å when the tensor orientation is unknown (Fig. 8b, c). This distance accuracy is still better than seen in the experimental data (Fig. 5) due to the absence of relayed transfer in these two-spin simulations. Further increasing the MAS rate to 60 kHz and comparing two rf fields of 50 kHz (f = 60%) (Fig. 8d) and 125 kHz (f = 24%) (Fig. 8e), we found reduced dependence on the CSA tensor orientation and an improved distance resolution of better than 0.5 Å. The polarization transfer rates are faster with weak rf pulses than with strong and short pulses, which can be understood as follows: under fast MAS, the CSA is increasingly averaged out and δ-pulse RFDR polarization transfer can no longer occur by the n = 0 rotational

### General Guidelines for Sample Preparation Strategies in HR-$\mu$MAS NMR-based Metabolomics of Microscopic Specimens

2.2. Sample Filling  The  sample  filling  procedure  can  be  considered  the  most  significant  step  in  the  sample  preparation.  This  is  because  of  its  extensive  manipulation  of  the  sample.  Unfortunately,  it  is  not  straightforward  to  fill  μg  specimens  into  a  tiny  rotor  (with  a  0.5  mm  inner  diameter).  It  should  comply with the following criteria: (1) a good sample homogeneity inside the μ‐rotor to achieve high  spectral resolution data (i.e., avoiding the presence of air bubbles). For example, the tiniest air pocket  can  worsen  the  spectral  resolution;  (2)  a  correct  sample  displacement  inside  the  μ‐rotor  for  maximum sensitivity detection; (3) a sufficient sample mass to achieve a good sensitivity; (4) a good  weight balance of the μ‐rotor to avoid spinning deficiency; and (5) a repeatable sampling procedure  for  data  reproducibility.  Subtle  deviations  in  all  these  criteria  could  affect  the  individual  spectral  data and diminish both the data repeatability and reproducibility. What follows are the details of  three strategies, each with different toolsets targeting different specimens.

### Cross-Correlated Relaxation of Dipolar Coupling and Chemical-Shift Anisotropy in Magic-Angle Spinning R 1ρ NMR Measurements: Application to Protein Backbone Dynamics Measurements

Introduction The structure of a protein is determined by a large number of individually weak interactions, which constantly re-shuffle at ambient temperatures. This dynamic exchange between different conformers is often important for functions such as binding or enzymatic turnover. NMR spectroscopy is very well suited to provide direct atomic-level access to the amplitudes and time scales of such motions. Whereas solution-state NMR is a well-established tool for such studies, magic-angle spinning (MAS) solid-state NMR (ssNMR) is rapidly establishing in studies of biomolecular dynamics, complementing its solution-state counterpart in cases where limited solubility or large size hamper the application of solution-state methods. In addition to opening new fields of applications, ssNMR also has theoretical advantages for determining dynamics, compared to solution-state NMR. In particular, the absence of overall tumbling motion enables the study of motions without “blind windows” of time scales, i.e. over all time scales from picoseconds to seconds, in contrast to its solution-state counterpart, which is severely challenged on the nanosecond to several-microseconds time scale. 1 In MAS ssNMR, motions can be studied through (i) their averaging effect on

### 100 kHz MAS Proton-Detected NMR Spectroscopy of Hepatitis B Virus Capsids

Keywords: solid-state NMR, fast MAS, proton detection, carbon detection, deuteration, hepatitis B virus, capsid, core protein INTRODUCTION Proton detection at 60 kHz magic-angle spinning (MAS) in 1.3 mm rotors in most cases requires protein deuteration ( Andreas et al., 2015 ). This often sacrifices expression yields in bacteria, and also reduces the number of proteins to be studied to those for which exchangeable protons, most importantly amide protons, can to a large extent be back exchanged from 2 H to 1 H. Compared to 60 kHz, MAS frequencies of 100 kHz further average 1 H dipolar interactions by a factor of ∼0.6 ( Penzel et al., 2019 ), improving resolution in fully-protonated systems and allowing to resolve the resonances of small proteins ( Cala-De Paepe et al., 2017; Lakomek et al., 2017; Schubeis et al., 2018 ). Still, comparison of 1 H linewidths on protonated and deuterated proteins has shown that