7200 gc qtof

Aliquots of 50–80 mg frozen leaf material were extracted with a mixture of chloroform-methanol-water for analysis of sugars, organic acids and amino acids by gas chromatography-mass-spectrometry (GC-MS) according to Fiehn et al. (2000) (link) using a 7200 GC-QTOF (Agilent, Milford, CT, United States). Peak integration was conducted with MassHunter Software from Agilent. For relative quantification, metabolite peak areas were normalized to the amount of extracted plant material and the peak area of the internal standard ribitol added to the extraction solution.

All GC-MS analysis was performed with an Agilent 7200 GC-QTOF and an Agilent 7693A automatic liquid sampler. Dried samples were suspended in 40 µL of a 40 mg/mL O-methoxylamine hydrochloride (MOX) (MP Bio #155405) in dry pyridine (EMD Millipore #PX2012-7) and incubated for 1 hr at 37°C in a sand bath. 25 µL of this solution was added to auto sampler vials. 60 µL of N-methyl-N-trimethylsilyltrifluoracetamide (MSTFA with 1%TMCS, Thermo #TS48913) was added automatically via the auto sampler and incubated for 30 min at 37°C. After incubation, samples were vortexed and 1 µL of the prepared sample was injected into the gas chromatograph inlet in the split mode with the inlet temperature held at 250°C. A 5:1 split ratio was used for analysis for the majority of metabolites. Any metabolites that saturated the instrument at the 5:1 split were analyzed at a 50:1 split ratio. The gas chromatograph had an initial temperature of 60°C for one minute followed by a 10 °C/min ramp to 325°C and a hold time of 10 min. A 30-meter Agilent Zorbax DB-5MS with 10 m Duraguard capillary column was employed for chromatographic separation. Helium was used as the carrier gas at a rate of 1 mL/min. Below is a description of the two-step derivatization process used to convert non-volatile metabolites to a volatile form amenable to GC-MS. Pyruvic acid is used here as an example.

Metabolites were extracted using 105 µL chloroform and 245 µL methanol. After incubation for 1 h at −20 °C we added 560 µL HPLC grade water twice. The samples were centrifuged for two minutes at high speed in a table top centrifuge at 4 °C and the aqueous phases were collected for the GC-MS measurements (in total about 1.3 mL).
For the metabolite analysis a gas chromatography – mass spectrometry (GC-MS) system (7200 GC-QTOF from Agilent) was used as described in^{53 (link)}. The data were analyzed with the Mass Hunter Software (Agilent). For absolute quantifications, we used five different dilutions of the standard mix (resulting in effective metabolite concentrations: 1 µM, 5 µM, 10 µM, 15 µM and 20 µM; Fig. S3) and calculated for each metabolite a standard curve which we used to determine the amount of the respective metabolite in our samples.