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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

Title Non-targeted metabolite profiling
Instrument LC: Acquity UPLC System (Waters, Milford, USA) , MS: QTOF Synapt G1 (Waters, UK)
Instrument Type
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.

Non-targeted metabolite profiling
The samples extracted with methanol were reconstituted in a mixture of methanol/deionized water/formic acid [75:24.85:0.15 (v/v/v)] and filtered through a 0.2-μm filter. Chromatographic separation was achieved on an Acquity UPLC System (Waters, Milford, USA) using a CSH C18 2.1 × 150 mm, 1.7 µm column maintained at 40 °C. The samples were injected (10 μl) and elution of compounds was performed at a flow rate of 0.3 ml min−1, the gradient comprised mobile phase A: deionized water containing 0.1% formic acid; and mobile phase B: acetonitrile containing 0.15% formic acid. The gradient program was isocratic for the first 30 s, then a linear gradient increase from 30 to 80% of solvent B at 6 min, 100% of B at 24.9 min, and hold for 1 min for column washing and 4 min at 30% of solvent B for column re-equilibration. The mass spectrometer comprised an orthogonal QTOF Synapt G1 (Waters, UK) operated under the following conditions: positive electrospray ionization mode, capillary voltage at 3.0 kV, cone voltage 45 V, extractor voltage 4.0 V, source and desolvation temperature were 150 and 350 °C, respectively. Cone and desolvation gas flow was nitrogen at a flow rate of 20 l h−1 and 700 l min−1. Leucine-enkephalin (M + H)⊕ = 556.2771 was infused at a flow rate of 5 μl min−1 at concentration of 2 ng ml−1 during acquisition as internal mass calibrant. MS data were acquired on continuum mode and processed with MassLynx (version 4.1, Waters, USA) and Progenesis QI for small molecules (Nonlinear Dynamics version 2.3, Waters, USA) software, using Progenesis MetaScope Database, with Biomolecules, LipidsMAPS, and LipidBlast as searching parameters, accepting precursor tolerance of 10 ppm for putative identifications.


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