Shim pack uflc

The tubers sliced to 1 mm thickness was crushed using a mixer mill (MM400, Retsch) with a zirconia bead for 1.5 min at 30 Hz. 100 mg flesh powder was extracted overnight at 4 °C with 1.0 ml 70% aqueous methanol. The sample extracts were analyzed using an LC-ESI-MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30A, MS, Applied Biosystems 6500 Q TRAP). The metabolomics approach was according to Yan et al. [67 (link)] with some modification.

After sample treatment and collection, a widely targeted metabolomics analysis was conducted at MetWare Biotechnology Co. Ltd (Wuhan, China) on the basis of a previously described method [12 ]. In brief, samples were ground into powder and then 100 mg powder was extracted in 1.0 mL of 70% aqueous methanol overnight at 4°C. Next, the extract was absorbed and filtered for further analysis using a UPLC–ESI–MS/MS system. This system was a combination of UPLC (Shim-pack UFLC, Shimadzu CBM30A system) and MS (Applied Biosystems 4500 Q TRAP). The conditions for the UPLC and MS were as described previously [12 ]. The MetWare database (MWDB) was used to identify the metabolites. The abundance of metabolites was calculated based on their peak areas. Metabolites with change in relative abundance >2-fold between −Pi and +Pi treatments, as well as variable importance in project (VIP) >1.0, were defined as DAMs. Lipidomic analysis was conducted at MetWare Biotechnology Co. Ltd (Wuhan, China) according to previously described methods [48 ].
Targeted determination of ATP, ADP, phosphoenolpyruvate (PEP), fructose 6-phosphate (F6P), and glucose 6-phosphate (G6P) using an LC–MS/MS system was performed at Shanghai Applied Protein Technology Co. Ltd (Shanghai, China) based on published methods [49 (link)]. Each treatment in this experiment had five biological replicates.

For the LC-MS/MS analysis, 1 volume of the thawed serum sample was mixed with 3 volumes of ice-cold methanol. The mixture was whirled and centrifuged twice, and the supernatant was obtained. The UPLC-ESI-MS/MS system (UPLC, Shim-pack UFLC SHIMADZU CBM A, Shimadzu Company, Japan; MS, QTRAP^®, SCIEX Company, USA) was employed. The analytical conditions were as follows: column temperature, 40 °C; flow rate, 0.4 mL/min; injection volume, 2 μL; solvent system, water (containing 0.04% acetic acid) and acetonitrile (containing 0.04% acetic acid); gradient program, 95:5 v/v at 0 min, 5:95 v/v at 11.0 min, 5:95 v/v at 12.0 min, 95:5 v/v at 12.1 min, and 95:5 v/v at 14.0 min. The metabolites were identified based on the standard data of Metware (Wuhan, China) and quantified according to their peak area.

The sample preparation and metabolomics analysis were performed as previously described^{49 (link)–51 (link)}. The sample extracts were analyzed by an LC–ESI–MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30A system, and MS, Applied Biosystems 6500 Q TRAP). The analytical conditions were performed according to Wang et al. (2018)^{50 ,51 (link)}. LIT (linear ion trap) and triple quadrupole (QQQ) scans were captured via triple quadrupole-linear ion trap mass spectrometer (Q TRAP), API 6500 Q TRAP LC/MS/MS System. The mass spectrometry conditions were performed according to Wang et al. (2018)^{50 ,51 (link)}.

Sample extracts were analysed using an LC-ESI-MS/MS system (UPLC, Shim-pack UFLC Shimadzu CBM A system, https://www.shimadzu.com/; MS, QTRAP® System, https://sciex.com/). Linear Ion Trap (LIT) and triple quadrupole (QQQ) scans were acquired on a QQQ-linear ion trap mass spectrometer QTRAP® LC-MS/MS system equipped with an electrospray ionization (ESI). The turbo ion-spray interface operates in positive and negative ion mode and is controlled by Analyst 1.6.3 software (Sciex). The ESI source operation parameters were as follows: source temperature 500 °C; ion spray voltage (IS) 5500 V (positive), −4500 V (negative); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) pressure were set at 55, 60, and 25.0 psi, respectively; and the collision gas (CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 µmol/L polypropylene glycol solutions in QQQ and LIT modes. For each period, a specific set of MRM transitions were monitored depending on which metabolites eluted within this period.