5500 qtrap hybrid triple quadruple linear ion trap mass spectrometer

The metabolites in the cell lysates and tissues from mice were measured by LC-MS/MS (ACQUITY UPLC) in the laboratory of Prof. Yi Zhu at Tianjin Medical University as they previously described [26 (link)]. Briefly, 100 mg of tumour tissues, livers, or lungs were homogenized before lipid extraction. HUVECs were lysed by repeated freeze–thawing cycles and then pre-processed in methanol. After centrifugation, the supernatant was extracted by ethyl acetate twice, and then the upper organic phase was evaporated. The residue was then dissolved in 100 μl 30% acetonitrile. The resulting sample was then subjected to ultra-high-performance liquid chromatography (Waters, Milford, MA) with a 5500 QTRAP hybrid triple-quadruple linear ion trap mass spectrometer (AB Sciex, Foster City, USA) equipped with a Turbo Ion Spray electrospray ionization source.

Metabolomic analysis involved liquid chromatography–tandem mass spectrometry (LC–MS/MS) of metabolites as described previously (16 (link), 17 (link)). The methyl-tert-butyl ether (MTBE)-based method was selected to detect lipid levels in mouse plasma and hearts. Briefly, 200 µl plasma or 50 mg heart tissue was spiked with an internal standard mixture. Then, 400 µl 75% methanol was added and mixed. Subsequently, 1 ml MTBE was supplemented and incubated with the mixture for 1 h. Then, 250 µl of sterile water was added to induce phase separation. Finally, the upper organic phase was transferred to a new tube, and the water phase was extracted again. The organic phase was combined and then evaporated to dryness after centrifugation for 10 min at 12,000×g.
Target profiling of GPLs metabolites was performed using a 5500 QTRAP hybrid triple quadruple linear ion-trap mass spectrometer (AB Sciex, Foster City, CA, USA) equipped with a turbo ion-spray electrospray ionization source. For data analysis, we used Metaboanalyst 3.0 (http://www.metaboanalyst.ca). Missing values were imputed with half of the minimum positive value, and data were log-transformed and autoscaled before analysis.