Quadrupole tof ms 5600 triple tof plus

For metabolic flux experiments, ¹³C-palmitate tracing was conducted using LC/MS at LipidALL Technologies. Cells (1 × 10⁷) were incubated for 8 h at 37 °C in RPMI-1640 medium with 10% FBS. For ¹³C-palmitate incorporation in metabolites, cells were incubated in growth medium with 50 μM [U-¹³C] palmitate for 24 h. Then, cells were quickly washed with 1×PBS and fixed with pre-cooled methanol (HPLC-MS grade, Millipore) for 30 min at −80 °C. The extraction method was referenced to a previous paper, but with modification [52 (link)]. Samples were incubated for 30 min at 1500 rpm and 4 °C and then centrifuged for 10 min at 12,000 rpm and 4 °C. The supernatant fractions were placed into clean 1.5-ml centrifuge tubes and dried using a SpeedVac. The dried extracts were dissolved with 50% acetonitrile in water and the upper layer liquids were collected for LC–MS analysis. The InfinityLab Poroshell 120 HILIC-Z column (2.1 mm × 50 mm, 2.7 μm, Agilent Technologies, Germany) was used in this study. Ultra-performance Liquid Chromatography (Agilent 1290 II, Agilent Technologies, Germany) coupled to the Quadrupole-TOF MS 5600 Triple TOF Plus, AB SCIEX, Singapore) was used to acquire metabolome data.

Untargeted metabolomics was conducted by LipidALL Technologies as previously described (Song et al. 2020 (link)). Metabolites were separated on an ACQUITY UPLC HSS T3 1.8 μm, 2.1 × 100 mm column (Waters, Dublin, Ireland) using ultra-performance liquid chromatography (Agilent 1290 II, Agilent Technologies, Germany) and analyzed on a Quadrupole-TOF MS (5600 Triple TOF Plus, AB SCIEX, Singapore). The MS parameters for detection were: ESI source voltage − 4.5 kV; vaporizer temperature, 500℃; drying gas (N₂) pressure, 50 psi; nebulizer gas (N₂) pressure, 50 psi; curtain gas (N₂) pressure, 35 psi; the scan range was m/z 60–800. The information-dependent acquisition mode was used for MS/MS analyses of the metabolites. The collision energy was set at 35 ± 15 eV and the data acquisition and processing were performed using the Analyst® TF 1.7.1 Software (AB Sciex, Concord, ON, Canada). All detected ions were extracted using MarkerView 1.3 (AB Sciex, Concord, ON, Canada) into Excel in the format of a two-dimensional matrix, including mass to charge ratio (m/z), retention time, and peak areas. Then, the isotopic peaks were filtered. The MS/MS data were extracted, and a comparison was performed with the Metabolites database (AB Sciex, Concord, ON, Canada), HMDB, METLIN using the PeakView 2.2 (AB Sciex, Concord, ON, Canada), and the standard references were used for annotating the ion ID.