Model 5975c ms

Cytosolic free amino acids from cells or tissue samples were extracted using 0.4 M ice-cold perchloric acid (PCA) [18 (link)]. Cellular protein were hydrolyzed in 6N HCl under vacuum. The amino acids were purified by cation-exchange chromatography [54 (link)]. Amino acids were converted to heptafluorobutyryl-propyl ester derivatives [28 (link)] and separated on an HP-5MS column (30 m × 0.25 nm). Isotopic enrichment was determined in electron capture negative ionization mode by gas chromatography-mass spectrometry (GC-MS) using a model 6890 GC and model 5975C MS (Agilent, Palo Alto, CA, USA), as described previously [24 (link),25 (link),31 (link)].
Genomic DNA was isolated and purified, as described previously [24 (link),25 (link)]. The DNA samples were dried and hydrolyzed in formic acid under vacuum then derivatized by N, O- Bis- [trimethylsilyl] trifluoroacetamide 1% trimethyl-chlorosilane and acetonitrile. Isotopic enrichments of ¹³C₁-serine in deoxyadenylate (dAMP, dA) and deoxyguanylate (dGMP, dG), and deoxythymidine (dTMP, dT), and deoxymethyl-cytosine (mC) were determined in positive ionization mode by GC/MS, as described previously [24 (link),25 (link),31 (link)].

Plasma and cytosolic free amino acids from cells or tissue samples were extracted using 0.4 M ice-cold perchloric acid (PCA) [59 (link),61 (link)]. Cellular proteins were hydrolyzed in 6 N HCl under a vacuum. The amino acids were purified by cation-exchange chromatography, converted to heptafluorobutyryl-propyl ester derivatives, and separated on an HP-5MS column (30 m × 0.25 nm). Isotopic enrichment was determined in electron capture negative ionization mode by gas chromatography–mass spectrometry (GC-MS) using a model 6890 GC and model 5975C MS (Agilent, Palo Alto, CA, USA), as described previously [54 (link),62 (link)].

O. maius was grown at 25°C in plastic (6 replicates) and glass (3 replicates) petri plates containing MS medium covered with cellophane membranes. VOCs were collected in the cultures headspace after 15 and 30 days from fungal inoculum. Control plates without mycelium were sampled for background correction. VOCs were collected for 6 h from sealed Petri dishes by headspace sorptive extraction using the stir bar sorptive extraction method with Gerstel Twisters (Gerstel GmbH & Co. KG, Mülheim an der Ruhr, Germany) as described in [33 (link)]. The samples were analysed with a thermo-desorption unit (Gerstel GmbH & Co) coupled to a gas chromatograph-mass spectrometer (GC-MS; GC model: 7890A; MS model: 5975C; Agilent Technologies, Santa Clara, CA, USA) as described in [34 (link)]. The chromatograms were analyzed by the enhanced ChemStation software (MSD ChemStation E.02.01.1177, 1989–2010 Agilent Technologies, Santa Clara, CA, USA). The TIC (Total Inorganic Carbon) of each VOC in the final dataset was recalculated from the absolute abundance of the first representative m/z to eliminate noise. The calibration was done as described in [34 (link)]. The emission rates were calculated on fungal mycelium area (pmol cm^-2 h^-1) bases.