Ontology highlight
ABSTRACT:
PROVIDER: PRJEB15896 | ENA |
REPOSITORIES: ENA
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Project description:Viscum album is known for its special mode of cellular respiration. It lacks the mitochondrial NADH dehydrogenase complex (complex I of the respiratory chain) and has restricted capacities to generate mitochondrial adenosine triphosphate (ATP). We here present an investigation of the V. album energy metabolism taking place in mitochondria. Mitochondria were purified from young V. album leaves and membrane bound protein complexes characterized by Blue native polyacrylamide gel electrophoresis as well as by the complexome profiling approach. Proteins were systematically identified by label-free quantitative shotgun proteomics.
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Project description:Viscum album is known for its special mode of cellular respiration. It lacks the mitochondrial NADH dehydrogenase complex (complex I of the respiratory chain) and has restricted capacities to generate mitochondrial adenosine triphosphate (ATP). We here present an investigation of the V. album energy metabolism taking place in the chloroplasts. Thylakoids were purified from young V. album leaves and membrane bound protein complexes characterized by Blue native polyacrylamide gel electrophoresis as well as by the complexome profiling approach. Proteins were systematically identified by label-free quantitative shotgun proteomics.
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Project description:Samples:
The plants were harvested from 5 different host threes: Abies alba, Malus domestica, Pinis sylvestris, Quercus sp., Ulmus carpinifolia. Samples were harvested in summer and winter seasons.V. album MT was prepared from fresh plant material according to previous methodology described by our group (Anvisa, 2011; Holandino et al., 2020). The fresh material was fragmented into parts smaller than 5 cm and submitted to an ethanolic maceration process for 21 days at room temperature shaking twice a day. The final alcoholic content ranged from 40 to 50% v/v. The mother tinctures were coded according to the respective host tree and the harvest season, as following: Viscum album subsp. abietis from Abies alba in summer (A-17) or winter (A-18), Viscum album subsp. album from Malus domestica in summer (M-17) or winter (M-18), Viscum album subsp. album from Quercus sp. in summer (Q-17) or winter (Q-18), Viscum album subsp. album from Ulmus carpinifolia in summer (U-17) or winter (U-18), Viscum album subsp. austriacum from Pinus sylvestris in summer (P-17) or winter (P-18).
2) Viscum album mother tinctures analysis by liquid chromatography coupled to high-resolution mass spectrometry:
Initially, 1.0 g of mother tincture was weighed and transferred to a 5 mL volumetric flask where a solution 9:1 acetonitrile/water with 0.1% v/v formic acid was added. After homogenization, the solution was centrifuged at 7,056 g for 15 minutes and then 200 microliters of each supernatant were removed for analysis in a DionexUltiMate 3000 liquid Chromatography coupled to a hybrid Quadrupole-Orbitrap high-resolution mass spectrometer (Thermo Q-Exactive Plus) equipped with an electrospray ionization source (ESI). Pooled quality control samples (QC) containing 10 microliters of each MT were also prepared. Chromatography separation was performed in a reversed-phase column (Hypersil Gold C18, 100 mm x 2.1 mm x 3.0 micrometers; Thermo Fisher Scientific). Water with 0.1% v/v of formic acid (A) and acetonitrile with 0.1% v/v of formic acid (B) were used as the mobile phases in an elution gradient of: (i) 0-1 min, 10% B; (ii) 1-16 min, 10-95% B; iii) 16-18 min, 95% B; (iv) 18-18.1 min, 95-10% B; (v) 18.1-22 min 10% B. The flow rate was 0.350 microliters/min and the injection volume 5 microliters. Mass spectra were acquired simultaneously in negative and positive ionization modes in the m/z range of 100-1000 at a resolution of 35k (FWHM) followed by sequential mass spectrometry (MS/MS) experiments ddMS2 top 3 (where the 3 most intense ions of each scan were fragmented automatically). The mass spectrometry conditions were the following: spray voltage 3.9 kV for ESI (+) or 3.6kV for ESI (-), ion transfer capillary 300 C, sheath, and auxiliary gases 45 and 15 arbitrary units, respectively. Data were analyzed by the Xcalibur 2.0.7 program (Thermo Scientific, Bremen, Germany) (Holandino et al., 2020).
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