Quantitative Metabolomic Analysis of NRK-49F Cells

Biological triplicate 10 cm² dishes were used to cultivate NRK-49F cells in full culture medium. Metabolites were extracted using 1 mL of ice-cold 80% methanol on dry ice. Subsequently, the samples were centrifuged at 14,000 rpm for 5 min. To ensure thorough extraction, the cell pellets were subjected to an additional extraction with 0.5 mL of 80% methanol. For accurate protein quantitation, the cell pellets were dissolved in an 8 M urea solution. The supernatant obtained from the metabolite extraction was desiccated into a pellet using SpeedVac from Eppendorf (Hamburg, Germany), using a heat-free technique. Before analysis, the dried pellets were re-suspended in 20 μL of HPLC-grade water in preparation for mass spectrometry, as described before [31 (link)]. A volume of 5–7 μL of the resulting resuspension was injected and subjected to analysis using a cutting-edge hybrid 6500 QTRAP triple quadrupole mass spectrometer from AB/SCIEX (Framingham, MA, USA), which was coupled to a Prominence UFLC HPLC system from Shimadzu (Kyoto, Japan). The analysis was carried out through selected reaction monitoring (SRM), targeting a comprehensive set of 298 endogenous water-soluble metabolites, enabling a thorough examination of the steady-state characteristics of the samples. The data have been deposited to MetaboLights (MTBLS8281) [32 (link)].