6890 gas chromatograph

All GC-MS analysis was performed with a Waters GCT Premier mass spectrometer fitted with an Agilent 6890 gas chromatograph and a Gerstel MPS2 autosampler. Dried samples were suspended in 40 µL of 40 mg/mL O-methoxylamine hydrochloride (MOX) in pyridine and incubated for one hour at 30°C. Twenty-five µL of this solution was transferred to autosampler vials. Ten µL of N-methyl-N-trimethylsilyltrifluoracetamide (MSTFA) was added automatically via the autosampler and incubated for 60 min at 37°C with shaking. After incubation, 3 µL of a fatty acid methyl ester standard was added via the autosampler then 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. The gas chromatograph had an initial temperature of 95°C for one minute followed by a 40°C/min ramp to 110°C and a hold time of 2 min. This was followed by a second 5°C/min ramp to 250°C, a third ramp to 350°C, then a final hold time of 3 min. A 30 m Phenomex ZB5-5 MSi column with a 5 m long guard column was employed for chromatographic separation. Helium was used as the carrier gas at 1 mL/min.

All GC-MS analysis was performed with a Waters GCT Premier mass spectrometer fitted with an Agilent 6890 gas chromatograph and a Gerstel MPS2 autosampler. Dried samples were suspended in 40 μl of a 40 mg/ml O-methoxylamine hydrochloride (MOX) in pyridine and incubated for one h at 30°C. To autosampler vials was added 25 μl of this solution. Ten μl of N-methyl-N-trimethylsilyltrifluoracetamide (MSTFA) was added automatically via the autosampler and incubated for 60 min at 37°C with shaking. After incubation, 3 μl of a fatty acid methyl ester standard solution was added via the autosampler then 1 μl of the prepared sample was injected to the gas chromatograph inlet in the split mode with the inlet temperature held at 250°C. A 10:1 split ratio was used for analysis. The gas chromatograph had an initial temperature of 95°C for one min followed by a 40°C/min ramp to 110°C and a hold time of 2 min. This was followed by a second 5°C/min ramp to 250°C, a third ramp to 350°C, then a final hold time of 3 min. A 30 m Phenomex ZB5-5 MSi column with a 5 m long guard column was employed for chromatographic separation. Helium was used as the carrier gas at 1 ml/min. Due to the high amounts of several metabolites including valine, leucine, isoleucine, proline, phosphate and inositol the samples were analyzed once more at a 10 fold dilution.

GC-MS metabolomic analysis was performed by Matabolon INC from our in vivo study, in which Phf2 or GFP were overexpressed in the liver of C57BL/6J mice for 3 weeks (15 mice per group). Metabolites were extracted by 80% methanol at −20 °C and dried by vacuum centrifugation. GC-MS analysis was performed with a Waters GCT Premier mass spectrometer fitted with an Agilent 6890 gas chromatograph and a Gerstel MPS2 autosampler. Data were collected using MassLynx 4.1 software (Waters). Metabolites were identified and their peak area was recorded using QuanLynx. Data were normalized for extraction efficiency and analytical variation by mean centering the area of D4-succinate.