Targetlynx application manager of masslynx 4

Data processing was performed in the TargetLynx application manager of MassLynx 4.1 (Waters). Absolute quantification was performed by interpolation from calibration curves prepared by serial dilutions of analytical grade standards (Sigma-Aldrich). Calibration curves were calculated by least-squares regression with 1/x weighting, and all response factors of both standards and biological extracts were corrected by the response factor of the corresponding U¹³C(¹⁵N)-isotopologue. Extract concentrations were corrected for dilutions and concentrations performed during sample preparation, and normalized to measured cell density (cells/L) or total DW (g/L) in cell lines and microorganisms, respectively. Intracellular metabolite concentrations were calculated from experimental cell volumes (pL) of cell lines and estimated using literature values for specific cell volume (L/g) of microorganisms (Table 2).

Downstream data processing was performed in TargetLynx application manager of MassLynx 4.1 (Waters). Ion abundances were normalized to total ion abundance in DU145 and HEK293 extracts. Metabolite levels in HAP1, JJN3, RPMI 8226, MC/CAR, HL60, NB4, and primary monocyte extracts were absolutely quantified by interpolation from calibration curves prepared by serial dilutions of analytical grade standards (Sigma-Aldrich) calculated by least squares regression. Response factors of the analytical standards and biological extracts were corrected by the response factor of their corresponding U¹³C-labeled isotopologue spiked into the samples. Extract concentrations were then normalized to experimental cell density at the time of sampling, measured on a MoxiZ cell counter with type S cassettes (Orflo Technologies).