NMR Spectroscopy of Biological Samples

Samples were resuspended in 0.6 ml NMR buffer consisting of 0.1 M phosphate buffer pH 7.4, 1 mM trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP), and 0.55 ml was transferred to a 5 mm NMR tube. Spectra were acquired on a Bruker Avance DRX600 NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany), with 1H frequency of 600 MHz, and a 5 mm inverse probe. Samples were introduced with an automatic sampler and spectra were acquired following the procedure described by Beckonert et al [15 (link)]. Briefly, a one- dimensional NOESY sequence was used for water suppression; data were acquired into 64 K data points over a spectral width of 12 KHz, with 8 dummy scans and 512 scans per sample. In addition, 2D ¹H-¹H COSY were acquired. Data were acquired into 512 × 4096 data points covering 6 × 6 KHz, with 3 scans for each increment.
Spectra were processed in iNMR 2.6.3 (Nucleomatica, Molfetta, Italy). Fourier transform of the free-induction decay was applied with a line broadening of 0.5 Hz. Spectra were manually phased and automated first order baseline correction was applied. Metabolite assignments were based on Tredwell et al [14 (link)]. Metabolite concentrations from 2D spectra, relative to the internal standard TSP were calculated using rNMR [16 (link)], and data were normalised by the probabilistic quotient normalisation method described by Dieterle et al [17 (link)].