Multiquant v2

Briefly, 5 μL of each sample was injected and analyzed using a hybrid 5500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC system (Shimadzu) using an Amide HILIC column (Waters) and analyzed with selected reaction monitoring (SRM) with positive/negative polarity switching. Peak areas from the total ion current for each of 134 metabolite SRM transition were integrated using MultiQuant v2.1 software (AB/SCIEX). Relative metabolite abundance was provided as integrated peak intensities.

A hybrid QTRAP 5500 triple quadrupole mass spectrometer (AB SCIEX), which is coupled to a Prominence UFLC HPLC system (Shimadzu), was used. First, each sample was resuspended in 20 μl high-performance liquid chromatography (HPLC)-grade water, then 5–7 μl of it was injected into the spectrometer. The selected reaction monitoring (SRM) method was used for steady-state analyses with 262 endogenous water-soluble metabolites with a dwell time of 3 ms for each SRM, and a total cycle time of 1.55 s, with 10–14 data points acquired for each metabolite. Voltages used for the positive and negative ion modes were +4950 and −4500 V, respectively. Hydrophilic interaction chromatography using a 4.6 mm × 10 cm Amide XBridge column (Waters) at 400 μl/min was used to deliver samples to the spectrometer. Following gradient sequence were used during the measurement: 85% buffer B (HPLC grade acetonitrile) to 42% B from 0 to 5 min, followed by 42% B to 0% B from 5 to 16 min, with no gradient from 16 to 24 min, and finally, gradients were run from 0% B to 85% B from 24 to 25 min, and 85% B was held for 7 min to re-equilibrate the column. Buffer A comprised 20 mM ammonium hydroxide/20 mM ammonium acetate (pH 9.0) in 95:5 water: acetonitrile. MultiQuant v2.1 software (AB SCIEX) was used to integrate total ion current peak.

Cell samples were resuspended in high-performance liquid chromatography (HPLC) grade water for mass spectrometry as described previously [14 (link)]. Briefly, the solutions were injected and analyzed using a hybrid 5500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu) via selected reaction monitoring (SRM) of a total of 274 unique endogenous water-soluble metabolites for steady-state analyses of samples. Some metabolites were targeted in both positive and negative ion mode for a total of 306 SRM transitions using positive/negative ion polarity switching. Peak areas from the total ion current for each metabolite SRM transition were integrated using MultiQuant v2.1 software (AB/SCIEX). Comprehensive metabolomic data analysis was performed using MetaboAnalyst 4.0 [104 (link)].

For steady state polar metabolite profiling, full RPMI media was refreshed on cells for two hours. Polar metabolites were extracted from cancer cells in vitro using 80% (v/v) aqueous methanol as described before^{75 (link)}. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed using a 6500 or 5500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu) with Amide HILIC chromatography (Waters). Data were acquired in selected reaction monitoring (SRM) mode using positive/negative ion polarity switching for steady-state polar profiling of greater than 260 molecules. Peak areas from the total ion current for each metabolite SRM transition were integrated using MultiQuant v2.0 software (AB/SCIEX). The original data were normalized to protein concentration determined by BCA assay.