Abstract
The low sensitivity and thus need for large sample volume is one of the major drawbacks of Nuclear Magnetic Resonance (NMR) spectroscopy. This is especially problematic for performing rich metabolic profiling of scarce samples such as whole cells or living organisms. This study evaluates a 1H HR-MAS approach for metabolic profiling of small volumes (250 nl) of whole cells. We have applied an emerging micro-NMR technology, high-resolution magic-angle coil spinning (HR-MACS), to study whole Saccharomyces cervisiae cells. We find that high-resolution high-sensitivity spectra can be obtained with only 19 million cells and, as a demonstration of the metabolic profiling potential, we perform two independent metabolomics studies identifying the significant metabolites associated with osmotic stress and aging.
Highlights
1H nuclear magnetic resonance (NMR) spectroscopy has gained recognition as a key analytical technique for metabolic profiling of complex bio-systems (Dunn and Ellis, 2005)
A total of 22 metabolites have been identified from the two studies and are summarized in Table S1 of the supporting information (SI). The capability of such rich-profiling from a sub-microliter sample volume (250 nl) is owed to the fact that high-resolution magic-angle coil spinning (HR-MACS) offers a 4.8-fold sensitivity enhancement in signal-to-noise (SNR) per-unit-mass compared to the coupled high-resolution magic-angle spinning (HR-MAS) probe. This enhancement factor has been calculated based on BH1 RMACS/BH1 RMAS at a given radio frequency input power (Hoult, 2000), where the B1 field can be www.frontiersin.org μNMR whole cell metabolic profiling determined from a standard nutation experiment
Such high SNR should allow for further reductions in sample size, or in signal-averaging for samples which are prone to rapid decay
Summary
The observed signal intensities in a typical 1H NMR spectrum provide a direct comparison of the metabolite contents in the samples without the need to construct calibration curves for every analyte, which is often the case for other analytical techniques For these reasons, 1H NMR spectroscopy is widely used in the study of metabolomes, offering a robust tool for rich-metabolic profiling (Reo, 2002). Many samples of interest are highly complex bio-mixtures (e.g., biopsies, whole living cells and organisms) and the heterogeneity in the magnetic susceptibility over the sample volume results in broadening of the observed NMR resonances; significantly reducing the ability to identify and quantify the metabolic content using traditional high-resolution NMR techniques. Palomino-Schätzlein et al have optimized a HR-MAS protocol for the study of whole cells using abundant Saccharomyces cervisiae cells and reported similar metabolic profiles to those obtained with high-resolution NMR of cell extracts (Palomino-Schätzlein et al, 2013)
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