High Resolution-Magic-Angle Spinning NMR Spectroscopy for Metabolic Phenotyping of Caenorhabditis elegans

Analysis of model organisms, such as the submillimeter-size Caenorhabditis elegans, plays a central role in understanding biological functions across species and in characterizing phenotypes associated with genetic mutations. In recent years, metabolic phenotyping studies of C. elegans based on H high-resolution magic-angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy have relied on the observation of large populations of nematodes, requiring labor-intensive sample preparation that considerably limits high-throughput characterization of C. elegans. In this work, we open new platforms for metabolic phenotyping of C. elegans mutants. We determine rich metabolic profiles (31 metabolites identified) from samples of 12 individuals using a H NMR microprobe featuring high-resolution magic-angle coil spinning (HR-MACS), a simple conversion of a standard HR-MAS probe to μHR-MAS. In addition, we characterize the metabolic variations between two different strains of C. elegans (wild-type vs slcf-1 mutant). We also acquire a NMR spectrum of a single C. elegans worm at 23.5 T. This study represents the first example of a metabolomic investigation carried out on a small number of submillimeter-size organisms, demonstrating the potential of NMR microtechnologies for metabolomics screening of small model organisms. T there are many analytical tools available for metabolomics studies, which investigate chemical pathways involving small molecules (metabolites) in biosystems such as biofluids and biopsies. Among them, H NMR spectroscopy offers a quantitative, nondestructive, and highthroughput analytical platform with minimal sample preparation and interference. Thus, NMR is widely used in metabolomics providing a robust metabolic profiling tool to discriminate samples of different biological status or origin. In particular, the use of H NMR spectroscopy has now emerged as a powerful analytical component for investigation of intact biopsies, thanks to the application of H-detected HR-MAS. This technique consists in spinning the sample rapidly at an angle of 54.74° with respect to the static magnetic field B0 to overcome magnetic field heterogeneities responsible for broad resonance lines that, in the absence of MAS, reduce the spectral information. Hence, H HR-MAS NMR is considered a near universal technique for providing unbiased and high-precision fingerprints of abundant metabolites in intact biological tissues, which has led toward a concept of real-time metabolic profiling of a surgical human biopsy in the clinical practice. In recent reports, Elena-Herrmann and co-workers have demonstrated its potential for metabotyping of intact Caenorhabditis elegans worms. These studies have paved a new platform for NMR-based metabolomics application to submillimeter-size organisms. However, since NMR is an inherently insensitive technique, HR-MAS analysis relies on large sample volume detection (typically corresponding to individual samples of more than 1000 worms). For this reason, the current HR-MAS NMR studies of C. elegans involve particularly labor-intensive sample preparation for the biologists. Meanwhile, sampling a large quantity of worms, which is necessary to record sufficient signal, implies that interindividual metabolic variability is averaged offering a global view of the metabolomics phenotype for a population of nematodes. Analysis of small numbers of worms would not only ease the sample preparation but could also allow for individual metabolic screening, opening new possibilities to characterize the phenotypic diversity within genotypes that is not achievable when working with large populations of worms. There are numerous approaches for improving NMR sensitivity that is represented by the signal-to-noise ratio SNR ∝ (1/Vnoise)[ω0(B1/i)], where vnoise is the total noise received Received: April 3, 2014 Accepted: May 22, 2014 Published: May 22, 2014 Article