Metabolite Extraction and Analysis Protocol

Cells were washed twice with phosphate buffered saline (Mediatech, Manassas, VA) (1 mM pH 7.4) before being scraped into 750 µL of ice-cold methanol/water (4/1 v/v). For cells treated with rapamycin, samples were spiked with internal standards (500 ng [¹³C₄]-succinate, 500 ng [¹³C₆]-citrate, 500 ng [¹³C₃]-pyruvate, 2 µg [¹³C₃]-lactate, 500 ng [¹³C₄,¹⁵N]-aspartate, 2 µg [¹³C₅,¹⁵N]-glutamate and 500 ng [¹³C₆]-glucose 6-phosphate). Samples were pulse-sonicated for 30 s with a probe tip sonicator and centrifuged at 16,000 × g for 10 min. The supernatant was transferred to two new tubes: 50 µL were transferred to one tube and diluted 5 times with 50 mM ammonium carbonate for direct analysis of the underivatized redox cycling metabolites (Figure 2) and 700 µL were transferred to one tube containing 300 µL of phenylhydrazine in water (3 mg/mL) for analysis of underivatized and derivatized metabolites (Figure 2). Derivatization was conducted by incubation at room temperature for 2 h before evaporation to dryness under nitrogen. 100 µL of water was used to re-suspend the samples. Injection volume was 5 µL in both methods. The phenylhydrazine-derivatized samples were run with gradient 1 and the underivatized samples were run with gradient 2.