Ab triple tof 6600

Chromatographic grade ammonium fluoride, 2-propanol, ammonium acetate, formic acid, ammonium hydroxide, ammonium formate, acetonitrile, and methyl tert-butyl ether (MTBE) were obtained from Sigma Aldrich. Mass spectrometry grade methanol was purchased from Thermo Fisher. The Q-Exactive Plus Orbitrap LC–MS/MS System was from Thermo Scientific. AB Triple TOF 6600 was purchased from AB Sciex (Massachusetts, USA). The Nexera LC-30A liquid chromatography system was from SHIMADZU. An ACQUITY UPLC BEH Amide column (1.7 μm, 2.1 mm × 100 mm) and ACQUITY UPLC CSH C18 column (1.7 µm, 2.1 mm × 100 mm) were obtained from Waters Corporation (Milford, MA, USA).

The untargeted metabolomics and lipidomics were carried out as previously described with minor modifications.^[92 (link)
^] All cells were maintained under standard conditions with DMEM medium containing 10% FBS for 48 h and then subjected to metabolomics analysis. 10⁷ cells were incubated with 800 µL cold methanol for protein removal and metabolite extraction after two PBS washes. The mixture was collected and centrifuged at 12 000 × g for 20 min, and the supernatant was further dried in a vacuum centrifuge. For LC‐MS analysis, samples were redissolved in 100 µL acetonitrile/water (1:1, v/v) solvent and centrifuged at 14 000 × g at 4 °C for 15 min. Then the supernatant was subjected to AB Triple TOF 6600 (AB SCIEX) and Q Exactive HF‐X (Thermo) for metabolomics and lipidomics, respectively (Shanghai Applied Protein Technology). For statistical analysis, the VIP value of each variable in the orthogonal partial least‐squares discriminant analysis model was calculated to indicate its contribution to the classification. The significantly differential metabolites were identified with the cutoff (VIP >1, P‐value <0.05). P‐values were generated by the two‐tailed Student's t‐test.

We performed LC-MS analysis with the Agilent 1290 Infinity UHPLC system interfaced with an AB Triple TOF 6600 (AB Sciex, Germany) and HILIC (150 × 4.6 mm, 5 μm) HPLC columns (Hi Chrom; Reading, UK). The HILIC mobile phase consisted of 25 mM ammonium acetate and 20 ammonia water in (A) HPLC-grade water and (B) acetonitrile. The solvent gradient was 95% B (0–0.5 min), 95–65% B (0.5–7 min), 65–40% B (7–8 min), and 40–95% B (9.1–12 min); it was then maintained at 95%, and the flow rate was 0.5 mL/min. Nitrogen sheath and auxiliary gas flow rates were maintained at 30 and 60 arbitrary units, respectively. The electrospray ionization interface was set to the positive/negative dual-polarity mode with a spray voltage of 5.5 kV and ion transfer capillary temperature of600°C. Full-scan data were obtained under a mass-to‐charge ratio (m/z) between 25 and 1000 amu for both ionization modes. To monitor stability and repeatability, quality control (QC) samples were prepared by pooling 10 µL of each sample. The QC samples were inserted after every five regular samples.