Acetyl-CoA Metabolite Sample Preparation and Extraction

HeLa cells were grown in Joklik media and split into four cultures to create biological replicates for both the ¹³C-glucose labeling and the D₃-acetate labeling. Time point samples were collected prior to transferring to ¹³C-glucose media or D₃-acetate media, as well at 0.5, 1, 4, 8, 12, 16, and 24 hours after transferring the cultures to their respective heavy media.

Cell pellets from each time point were resuspended in a cold (−80 °C) solution of 80% methanol/20% H₂O, and then incubated in a −80 °C freezer for 15 minutes. The suspension was centrifuged immediately at 3400 × g for 5 mins at 4 °C. After collecting the supernatant, the pellet was resuspended in 80% methanol/20% H₂O, and the previous two steps were repeated. The two supernatants were dried in a speed vacuum, resuspended in water, and combined. Insoluble debris was pelleted by centrifugation prior to analysis by mass spectrometry.