Muss hunter software

Derivatization was performed using automated sample prep WorkBench instrument (Agilent Technologies, Santa Clara, CA, USA). Dried polar metabolites were dissolved in 60 μL of 2% methoxyamine hydrochloride in pyridine (ThermoFisher) and held at 40 °C for 6 h. After the reaction, 90 μL of MSTFA (N-Methyl-N-(trimethylsilyl) trifluoroacetamid) was added, and samples were incubated at 60 °C for 1 h. Derivatized samples were analyzed by GC-MS using a DB-35MS column (30 m × 0.25 mm i.d. × 0.25 µm) installed in an Agilent 7890B gas chromatograph (GC) interfaced with an Agilent 7200 Accurate-Mass Quadrupole Time-of-Flight (QTOF) mass spectrometer (MS), operating under electron impact (EI) ionization at 70 eV. Samples (1 μL) were injected in a splitless mode at 250 °C, using helium as the carrier gas at a flow rate of 1 mL/min. The GC oven temperature was held at 100 °C for 2 min and increased to 325 °C at 10 °C/min. GC/MS data processing was performed using Agilent Muss Hunter software. Relative metabolites abundance was carried out after normalization to internal standard d27 Myristic acid and cell number and statistical analyses were performed using MetaboAnalyst 4.0 [32 (link)].

After 144 hours of growth, the metabolites were extracted and analyzed by gas chromatography-mass spectrometry (GC-MS) as previously described [19 (link)] and the spent media were collected and filtered. 120-μl of ice-cold acetonitrile was added to 40 μl of media and vortexed at 4°C for 2 minutes, and the mixture was incubated on ice for 5 minutes and centrifuged at maximum speed for 10 minutes. A 100-μl volume of the aqueous phase was collected in a new tube and evaporated under nitrogen flow at 37°C. Derivatization was performed as described [72 (link)]. A GC/MS analysis was performed as previously described. A 1-μl volume of sample was injected in a 1:10 split mode at 250°C. The GC was performed as previously described [72 (link)]. The GC/MS data processing and metabolite quantification were performed using Agilent Muss Hunter software.
The following metabolites were detected: glutamine, glutamate, aspartate, glycine, serine, alanine, trehalose, methionine, lysine, tyrosine, cysteine, ornithine, putrescine, glucose, pyruvate, lactate, citrate, succinate, fumarate, malate, acetyl-CoA. Raw data are provided in S4 File.