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High-resolution magic-angle-spinning NMR spectroscopy for metabolic profiling of intact tissues

Nature Protocols volume 5, pages 1019–1032 (2010)Cite this article

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Abstract

Metabolic profiling, metabolomic and metabonomic studies require robust study protocols for any large-scale comparisons and evaluations. Detailed methods for solution-state NMR spectroscopy have been summarized in an earlier protocol. This protocol details the analysis of intact tissue samples by means of high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy and we provide a detailed description of sample collection, preparation and analysis. Described here are 1H NMR spectroscopic techniques such as the standard one-dimensional, relaxation-edited, diffusion-edited and two-dimensional J-resolved pulse experiments, as well as one-dimensional 31P NMR spectroscopy. These are used to monitor different groups of metabolites, e.g., sugars, amino acids and osmolytes as well as larger molecules such as lipids, non-invasively. Through the use of NMR-based diffusion coefficient and relaxation times measurements, information on molecular compartmentation and mobility can be gleaned. The NMR methods are often combined with statistical analysis for further metabonomics analysis and biomarker identification. The standard acquisition time per sample is 8–10 min for a simple one-dimensional 1H NMR spectrum, giving access to metabolite information while retaining tissue integrity and hence allowing direct comparison with histopathology and MRI/MRS findings or the evaluation together with biofluid metabolic-profiling data.

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Figure 1: Details of an MAS NMR probe: the rotor containing the sample is positioned in the hole of the stator block at 54.7° in the NMR probe head.
Figure 2: Standard pulse 600 MHz 1H NMR spectra acquired from control mouse liver tissue (reproduced with permission from ref. 74):
Figure 3: Example of an 800 MHz 1H–1H TOCSY MAS NMR spectrum of rat renal cortex tissue from a bromoethanamine-treated rat (6 h post dose) for the assignment of 1H–1H connectivities of metabolite signals (reproduced with permission from ref. 30).
Figure 4: 1H–13C HMQC MAS NMR spectrum from normal rat renal outer cortex tissue, acquired at a spinning rate of 4,200 Hz for the assignment of 1H–13C connectivities of metabolite signals (reproduced with permission from ref. 29).
Figure 5: Two rotor designs allowing the positioning of either 65 μl or 12 μl of sample volume in a zirconia rotor.
Figure 6: HR-MAS NMR preparation tools and tissue sample.
Figure 7: Example of edited 600 MHz NMR spectra of the same control liver tissue sample (all were acquired with water peak suppression) (reproduced with permission from ref. 16).
Figure 8: Time-dependent changes in rat liver tissue after treatment with GalN.
Figure 9: 1H HR-MAS magnetic resonance spectra obtained from four different regions (labeled from a (mostly healthy) to d (highest degree of necrosis)) of a resected, untreated parietal glioblastoma multiforme (GBM).
Figure 10: Comparison of MRI, MRS and HR-MAS of prostate cancer.

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Acknowledgements

We thank our academic and industrial collaborators for helpful discussions in the formulation of this paper. Y. Wang acknowledges the financial support from the Chinese Academy of Science (KJCX2-YW-W11) and MOST (2009CB118804). The MRC Integrative Toxicology Training Partnership (ITTP) is acknowledged for financial support to M. Coen.

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  1. Department of Surgery and Cancer, Biomolecular Medicine, Faculty of Medicine, Imperial College London, South Kensington, London, UK

    Olaf Beckonert, Muireann Coen, Hector C Keun, Timothy M D Ebbels, Elaine Holmes, John C Lindon & Jeremy K Nicholson

  2. State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, The Chinese Academy of Sciences, Wuhan, China

    Yulan Wang

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O.B. and J.C.L. drafted the initial manuscript and all of the authors contributed to its final form.

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Beckonert, O., Coen, M., Keun, H. et al. High-resolution magic-angle-spinning NMR spectroscopy for metabolic profiling of intact tissues. Nat Protoc 5, 1019–1032 (2010). https://doi.org/10.1038/nprot.2010.45

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