GC-MS Analysis of Amino Acid Metabolism

LCL cells were placed in T‐25 flasks upright at 1 × 10⁶ cells per ml in 10 ml RPMI ± Asn per replicate. After 24 h, the media were collected, cleared by centrifugation, and stored frozen at −80°C until processing. The cells were washed with cold 0.9% saline, then flash frozen in liquid nitrogen, and stored at −80°C as well. Cell pellets and media samples were processed and analyzed by gas chromatography‐mass spectrometry (GC‐MS), as reported previously.²⁴, ³¹, ³² GC‐MS data acquisition was accomplished with a Thermo Scientific Single Quadrupole Mass Spectrometer (ISQ) and Gas Chromatograph (Trace 1310). Amino acid peak areas were processed with XCalibur Quan Browser software and normalized to the peak area of a DL‐norleucine internal standard. Asn concentrations were quantified with an external calibration curve.