Non-targeted metabolomics by 1H nuclear magnetic resonance spectroscopy; an interspecies comparison of milk between dromedary, giraffe and white rhinoceros with observations on blesbok

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Metabolomes of milk of dromedary, giraffe, white rhinoceros and blesbok

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Filter 1 ml milk samples with Amicon Ultra – 2mL centrifugal units with 10kDa membrane filters at 12000 x g for five minutes. Filter 600 μl serum at 4500 x g for 30 minutes in a swing-bucket centrifuge using the same membrane filters. To 540 μl of filtered serum add 60 μl NMR buffer solution (1,5 M potassium phosphate solution in deuterium oxide with internal standard trimethylsily1-2,2,3,3-tetradeuteropropionic acid {TSP}, pH 7.4). Vortex and transfer to a 5 mm NMR tube for analysis. NMR spectroscopy on a Bruker Avance III HD NMR spectrometer, equipped with a triple resonance inverse (TXI) 1H {15N, 13C} probe head and x, y, z gradient coils, at 500MHz. 1H spectra were acquired as 128 transients in 32K data points. The spectral width was 6002 Hz and an acquisition time of 2.72 seconds and receiver gain was set to 64. The sample temperature was maintained at 300K and the H2O resonance was pre-saturated by single-frequency irradiation during a relaxation delay of 4s with a 90° excitation pulse of 8μs. Shimming of the sample performed automatically on the deuterium signal. Fourier transformation and phase and baseline correction done automatically. Software for NMR processing: Bruker Topspin (V3.5). NMR spectral analysis, peak annotation and quantification using Bruker AMIX (V3.9.14). Phase and baseline distortions of transformed spectra corrected by using Topspin 3.2 (Bruker).

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