3400 uhplc system

To quantify metabolites of central carbon metabolism (CCM) in mouse liver, we used UPLC-MRM/MS, a Dionex 3400 UHPLC system (Dionex Sunnyvale, CA, USA) coupled to a 4000 QTRAP mass spectrometer (SCIEX, Framingham, MA, USA). Liver metabolites were extracted and quantified including those from tricarboxylic acid (TCA) cycle, glucose and selected sugar phosphates, and other phosphate-containing metabolites and nucleotides (Tables S4 and S5). Concentrations of the detected metabolites were calculated from internal linear-regression calibration curves.

Whole frozen plantaris muscles were sent to Creative Proteomics (Shirley, NY, USA) for the quantification of NADP, NADPH, and 6-phosphogluconate levels. Muscles were weighed and then homogenized in cold 80% aqueous MeOH using a MM 400 mill mixer 2 × 1 min. Samples were centrifuged at 21,130× g at 5 °C, and 400 µL of clear supernatant removed and mixed with 100 µL of a 13C10-GTP (internal standard) solution and 250 µL chloroform. The mixture was vortexed for 1 min and then centrifuged at 21,130× g at 5 °C for 10 min. The upper aqueous phase was removed and dried under a nitrogen gas flow. The residue was reconstituted in 200 µL of 50% acetonitrile. A 10-µL aliquot of each sample was injected into a HILIC (hydrophilic interaction liquid chromatography) column (2.1 × 150 mm, 1.7 µm) for gradient elution with ammonium buffer (mobile phase A) and acetonitrile (mobile phase B). The efficient gradient was from 75% B to 15% B in 12 min, with a flow rate of 300 µL/min and a column temperature of 30 °C. Metabolites were detected by UPLC-MRM/MS with (-) ion detection using a Dionex 3400 UHPLC system coupled to a 4000 QTRAP mass spectrometer.