Qtrap 5500 lc ms ms system

All metabolomic analyses were conducted under steady-state conditions. KPC cell lines were grown in appropriate growth media in six replicates in 10-cm plates. Two hours before metabolite collection, cells were incubated in fresh growth media. Accordingly, replicate cell lines were plated and grown in parallel in order to control for cell growth. Cell counts from the replicate plates were used to normalize metabolite readings. After incubation in fresh growth media, 4 mL of 80% methanol that was pre-chilled to −80 °C was added, and the cell plates were immediately transferred to −80 °C to incubate overnight. The cell lysate-methanol mixture was then scraped and transferred to conical tubes on dry ice and centrifuged at 5000× g for 5 min. The supernatant was collected, and the process was repeated two more times after resuspending the pellet in 500 µL of chilled 80% methanol, for a total volume of 5 mL. Samples were then completely dried via speed vacuum at 30 °C. Metabolites were analyzed using a 5500 QTRAP LC-MS/MS system (SCIEX, Framingham, MA, USA) via selected reaction monitoring (SRM). Mass spectrometric peak area integration was done using MultiQuant 2.0 software (SCIEX, Framingham, MA, USA) as previously described [12 (link),13 (link)].