Complete Protocol for Slow-Spinning High-Resolution Magic-Angle Spinning NMR Analysis of Fragile Tissues

Abstract

High-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) is an essential tool to characterize a variety of semisolid systems, including biological tissues, with virtually no sample preparation. The "non-destructive" nature of NMR is typically compromised, however, by the extreme centrifugal forces experienced under conventional HR-MAS frequencies of several kilohertz. These features limit the usefulness of current HR-MAS approaches for fragile samples. Here, we introduce a full protocol for acquiring high-quality HR-MAS NMR spectra of biological tissues at low spinning rates (down to a few hundred hertz). The protocol first consists of a carefully designed sample preparation, which yields spectra without significant spinning sidebands at low spinning frequency for several types of sample holders, including the standard disposable inserts classically used in HR-MAS NMR-based metabolomics. Suppression of broad spectral features is then achieved using a modified version of the recently introduced PROJECT experiment with added water suppression and rotor synchronization, which deposits limited power in the sample and which can be suitably rotor-synchronized at low spinning rates. The performance of the slow HR-MAS NMR procedure is demonstrated on conventional (liver tissue) and very delicate (fish eggs) samples, for which the slow-spinning conditions are shown to preserve the structural integrity and to minimize intercompartmental leaks of metabolites. Taken together, these results expand the applicability and reliability of HR-MAS NMR spectroscopy. These results have been obtained at 400 and 600 MHz and suggest that high-quality slow HR-MAS spectra can be expected at higher magnetic fields using the described protocol.

Recent Advances in NMR-Based Metabolomics

“…To overcome this issue, a protocol for acquiring the spectra at very low spinning speeds of a few hundred hertz was provided. 38 In this experiment, the suppression of broad spectral features is achieved using a modified version of the recently introduced PROJECT experiment with water suppression and rotor synchronization.…”

Hepatic metabolic effects of Curcuma longa extract supplement in high-fructose and saturated fat fed rats

“…This is achieved by spinning of the sample around inside the NMR probe at an angle of 54.7 ° with respect to the magnetic field at rotational frequency of a few kHz. In the case of fragile samples that could be damaged by rotation stress, protocols of sample handling have been described allowing for much slower spinning 13, 14 . HRMAS NMR has been successfully used to discover metabolic biomarkers to study in a variety of fields, notable examples being nutrition models and cancer discrimination 15, 16 .…”

Characterization of Brain Metabolism by Nuclear Magnetic Resonance

“…The net effect is spectra in which small molecules exhibit sharp resonances while more immobile macromolecules contribute to broad peaks underlying these resonances (Figure ). Typically, a sample volume of <100 μL is used and spinning rates are lower than those used in traditional MAS methods, with rates of up to 5 kHz or even lower for particularly fragile samples . For a well‐shimmed brain tissue sample, line widths approaching those seen in a solution state sample can be achieved for small molecule metabolites, typically about 0.005 PPM.…”

“…In practice, an inter‐echo time is chosen that experimentally gives the least obvious issues with J modulation for metabolites of interest. An alternative sequence, Periodic Refocusing of J Evolution by Coherence Transfer (PROJECT), initially proposed for use in solution NMR experiments (below), has successfully been demonstrated in low‐rotational rate HRMAS experiments on delicate tissues . It essentially introduces additional excitation pulses interspersed in the echo train and applied at a 90° phase offset relative to the original excitation pulse.…”