Abstract
Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique and has been widely used in metabolomics. However, the intrinsic low sensitivity of NMR prevents its applications to systems with limited sample availabilities. In this study, a new experimental approach is presented to analyze mass-scarce samples in limited volumes of less than 300 nL with simple handling. The sample is loaded into the glass capillary, and this capillary is then inserted into a Kel-F rotor. The experimental performance of the capillary-inserted rotor (capillary-insert) is investigated on an isotropic solution of sucrose by the use of a high-resolution micro-sized magic angle spinning (HRµMAS) probe. The acquired NMR signal’s sensitivity to a given sample amount is comparable or even higher in comparison to that recorded by the standard solution NMR probe. More importantly, this capillary-insert coupled with the HRµMAS probe allows in-depth studies of heterogeneous samples as the MAS removes the line broadening caused by the heterogeneity. The NMR analyses of mass-limited cultured neurospheres have been demonstrated, resulting in high quality spectra where numerous metabolites are unambiguously identified.
Highlights
Metabolomics is the qualitative and quantitative study of low weight molecules in biological systems, which enables a deeper understanding of the metabolic pathways of interest under defined conditions
The spectral broadening caused by the discontinuity of magnetic susceptibility within the sample can be removed by magic angle spinning (MAS), namely high-resolution MAS (HRMAS) or field-gradient MAS (FGMAS)
NL, the neurosphere count within this active volume is less than one unit. This suggests the possibility of individual specific analysis of neurosphere samples. With this new rotor design combined with a HRMACS is the modified μMAS probe (HRμMAS) probe, high-quality spectra of single neurospheres can be acquired for mass-limited samples
Summary
Metabolomics is the qualitative and quantitative study of low weight molecules (metabolites) in biological systems, which enables a deeper understanding of the metabolic pathways of interest under defined conditions. The advantages are: (i) higher radio frequency efficiency and optimal filling factor (the fraction of the sample with the detection coil volume), achieving higher sensitivity, and (ii) the removal of the magnetic susceptibility broadening from the μcoil and the sample heterogeneity, yielding high resolution up to 0.002 ppm [12] This technique managed to characterize the metabolic profiles of intact cells and whole tiny organisms. A further advancement for simplifying the labor work of HRMACS is the modified μMAS probe (HRμMAS), where no μcoil manipulation is required [16,17] In this experimental design, the use of (i) copper–aluminum as coil material for better matching with the magnetic susceptibility of air, and (ii) the Kel-F (polychlorotrifluoroethylene) rotor instead of zirconia, as well as the replacement of zirconia air-bearings inside the MAS stator by Vespel offered sufficient resolution for the study of metabolomic profiling of animal organs, such as liver and brain [17]. We will test the practical performance of the capillary-inserted Kel-F rotor (capillary-insert) coupled with the HRμMAS probe on the isotropic solution of sucrose, and investigate the feasibility of this setup for studying the NMR-based metabolomics for heterogeneous neurosphere samples with tiny quantities
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