Thermo q exactive focus mass spectrometer

After the samples were slowly thawed at 4°C in a salt-ice bath, 100 µl serum sample was precisely transferred by a pipettor to the 1.5 ml centrifuge tubes. Metabolites were extracted using a 400 µl methanol: water (4:1, v/v) solution with 0.02 mg/ml L-2-chlorophenylalanin as an internal standard, vortexed for 30 s, and extracted with low-temperature ultrasound for 30 min (5°C, 40 kHz). After extraction, the samples were left to stand at −20°C for 30 min. The sample was centrifuged (13000 rpm, 4°C, 15 min), and the supernatant was transferred to an UPLC-MS/MS injection vial for detection. Liquid chromatographic separations were performed on a Thermo Ultimate 3000 system (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) equipped with a Waters ACQUITY UPLC® HSS T3 column (150 × 2.1 mm, 1.8 μm). Mass spectrometry operations were performed on a Thermo Q Exactive Focus mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) with spray voltages of 3.8 kV in positive ion mode (ESI+) and −2.5 kV in negative ion mode (ESI−), respectively.
A pooled quality control (QC) sample was prepared by mixing equal volumes of all samples. Metabolomics data have been deposited to the EMBL-EBI MetaboLights database (DOI: 10.1093/nar/gkz1019, PMID:31691833) with the identifier MTBLS4245. The complete dataset can be accessed here https://www.ebi.ac.uk/metabolights/MTBLS4245.

Serum samples from 400 mg/kg ADe-treated HFD-fed mice, vehicle-treated HFD-fed mice, and vehicle-treated NCD-fed mice (n = 3) were randomly collected for lipidome analysis. The samples were analyzed via LC-MS/ESI-MSn using a Thermo Ultimate 3000 system equipped with an ACQUITY UPLC BEH C18 column and a Thermo Q Exactive Focus mass spectrometer (Thermo Fisher Scientific), as described in our previous study [23 (link)]. Significant statistical differences in the metabolites (p ≤ 0.05) and variable importance in projection values ≥1.0 were regarded as the standard values for differential lipids to filter out biomarkers.