Pi45950

Cells were plated in fresh media and incubated for 48 h. Protein quantification was performed using proxy wells for normalization. Norvaline was used as an internal standard. Citrate and lactate standards were used to generate a standard curve for quantification. Metabolite extraction was performed using HPLC-grade ethanol (Sigma Aldrich). Samples were derivatized with methoxamine (PI45950, Thermo Fisher Scientific) and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide with 1% tert-butyldimethylchlorosilane (Sigma Aldrich). Samples were analyzed by GC/MS using a HP-5MS Ultra Inert GC column (19091S-433UI, Agilent Technologies) installed in an Agilent 7890B gas chromatograph coupled to an Agilent 5977B mass spectrometer. Helium was used as the carrier gas. One microliter of sample was injected at 280°C. After injection, the GC oven was held at 60°C for 1 min. The oven ramped up to 320°C at 10°C/min and held for 9 min. The MS system operated under electron impact ionization mode at 70 eV, and the MS source and quadrupole were held at 230°C and 150°C, respectively. Peak abundance was determined by automated integration using MassHunter software (Agilent). Total abundance was normalized to the norvaline internal standard. Secretion rate was calculated by taking into account the specific growth rate, as determined by pre- and post-assay protein quantification.