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Sample Set Information

ID TSE1329
Title Funaria hygrometrica Hedw. elevated tolerance to D2O: its use for the production of highly deuterated metabolites.
Description The method introduced here to grow F. hygrometrica in high concentrations of D 2 O is an excellent alternative to produce highly deuterated metabolites with broad applications in metabolic studies. Our mass spectrometry experiments strongly indicate the successful incorporation of deuterium into organic compounds.. This approach also has limitations as D2O in high concentrations negatively affects the survival of most organisms. Here we report the moss Funaria hygrometrica as an unusual high tolerant to D2O in liquid culture. We found that this moss is able to grow in up to 90% D2O, a condition lethal for many eukaryotes. Mass spectrometric analyses of F. hygrometrica extracts showed a strong deuteration pattern. The ability to tolerate high concentrations of D2O together with the development of a rich molecular toolbox makes F. hygrometrica an ideal system for the production of valuable deuterated metabolites.
Authors Vergara, F., Itouga, M., Becerra, R.G., Hirai, M.Y., Ordaz-Ortiz, J.J., and Winkler, R.(2018)
Reference Planta (2018) 247: 405. DOI: 10.1007/s00425-017-2794-5

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Analytical Method Details Information

Instrument Accela LCQ Fleet Ion trap (Thermo Finnigan)
Instrument Type UPLC-QTOF-MS
Ionization ESI
Ion Mode positive
Description Plant metabolites extraction

After cell saturation was achieved F. hygrometrica cultures were transferred to plastic containers, flash-frozen with liquid nitrogen and subsequently freeze-dried. Dried plant material was ground with a Mixer Mill MM 301 (Retsch®) at 30 Hz for 15 s. Plant powder (20 mg of every sample) was extracted with 1.5 ml of HPLC grade methanol for 25 min in ultrasound bath at room temperature. Extracts were centrifuged at 15,870g for five min at room temperature and the supernatants were filtered using a 0.2 µm nylon syringe filter. Filtrates were subjected to UPLC–MSn analysis.

Chromatography/mass spectrometry conditions
Funaria hygrometrica extracts were analyzed using an ultra performance liquid chromatography electrospray mass spectrometry (UPLC–ESI–MSn) system Accela LCQ Fleet Ion trap (Thermo Finnigan). The compound mixture was separated on a Hypersil Gold C18 column (50 × 2.1 mm, 1.9 µm particle size). 10 µl were injected per sample. The mobile phase consisted of H2O with 0.1% (v/v) formic acid (solvent A) and methanol with 0.1% (v/v) formic acid (solvent B). The solvent gradient program was as follows: 30% B, 0–0.5 min; 30–80% B, 0.5–6 min; 80–100% B, 6–24.9 min; 100–30% B, 24.9–25 min; 30% B, 25–30 min. The flow rate was 400 µl min−1. Column oven temperature was maintained at 35 °C. Spectra were acquired in full scan mode 50–1000 m/z range, operating in positive mode; the scan time was 500 ms (3 micro-scans). The ESI source parameters were set as follows: capillary temperature 320 °C; capillary voltage 20 V; spray voltage 4.5 kV; tube lens 40 V; nitrogen sheath gas 25 arbitrary units (AU); auxiliary gas 15 AU. For the MSn analysis, collision energy (CID) was normalized to 35.


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