Cbm30a

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.

Nonvolatile compounds were identified using an LC-ESI-MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30A, Kyoto, Japan; MS, Applied Biosystems 6500 Q TRAP, Kyoto, Japan). For the HPLC conditions, the chromatographic column used was Waters ACQUITY UPLC HSS T3 C18 (2.1 mm × 100 mm, 1.8 µm, Waters Company, Milford, MA, USA). For the mobile phase, phase A was 0.04% acetic acid, and phase B was acetonitrile solution containing 0.04% acetic acid. The elution gradient of phase B was 0.00–11.00 min, 5–95%, maintained at 95%, 1.00 min; 11.00–12.10 min, 95–5%, and balanced at 5% until 15.00 min. The flow rate was 0.4 mL/min, the column temperature was 40 °C, and the injection volume was 2 μL.
For the mass spectrometry parameters, the electrospray ionization (ESI) temperature was 500 °C, the mass spectrum voltage was 5500 V, the curtain gas (CUR) was 25 psi, and the parameter of the collision-activated dissociation (CAD) was set to high. In the triple quadrupole, each ion pair was scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).

The freeze-dried fronds were grounded with zirconia beads by a mixer mill (MM 400, Retsch) at 30 Hz for 1.5 min. The powder (100 mg) was weighed and dissolved with 1.0 mL 70% methanol and extracted at 4°C overnight. After centrifugation at 10,000 g for 10 min, the sample extracts (supernatant) were collected and filtered by 0.22 μm pore size millipore filters, and then analyzed by an LC-MS/MS system (UPLC, Shim-pack UFLC SHIMADZU CBM30A, MS/MS Applied Biosystems 6500 QTRAP). Metabolites were identified by comparing the m/z values, the retention time (RT), and the fragmentation patterns with the standards in the database, and metabolites quantitate analysis was performed by MRM (multiple reaction monitoring) (Dong et al., 2019 (link)). QC samples were exported to analyze the sample repeatability under the same treatment. According to the result of OPLS-DA, the VIP (variable importance in the project) score was used to screen the significantly changed metabolites (SCMs). Differential metabolites with VIP ≥ 1, fold change ≥ 2, and fold change ≤ 0.5 were filtered as significantly changed metabolites. The MRM was carried out by Metware Biotechnology Co., Ltd. (Wuhan, China).

The sample extracts were analyzed using an Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI–MS/MS) system (HPLC, Shimpack UFLC Shimadzu CBM30A, Kyoto, Japan; MS, Applied Biosystems 6500 Q TRAP). The analytical conditions were as follows, HPLC: Column, Waters ACQUITY UPLC HSS T3 C18 (1.8 µm, 2.1 mm × 100 mm); Solvent system, water (0.04% acetic acid): Acetonitrile (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, 95:5 V/V at 15.0 min; flow rate, 0.40 mL/min; temperature, 40 °C; injection volume: 2 μL. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (Q TRAP)-MS. After each injection, the needle was rinsed with 600 μL of weak wash solution (water/methanol: 90:10) and 200 μL of strong wash solution (methanol/water: 90:10). The samples were maintained at 6 °C during analysis.

Quantitative analysis of palmitoyl carnitine was carried out as previously descripted.^[50] The fresh BAT tissue or brown adipocytes were sent to Metware Biotechnology Co., Ltd. (Wuhan, China) for LC–MS/MS analysis. The sample extracts were analyzed using a liquid chromatography electrospray tandem mass spectrometry (LC–ESI–MS/MS) system (ultra performance liquid chromatography, UPLC, Shim‐pack; ultrafast liquid chromatography, UFLC, SHIMADZU CBM30A; MS/MS, Applied Biosystems 6500 QTRAP). Palmitoyl carnitine was analyzed using scheduled multiple reaction monitoring.