Targeted Proteomics of Labeled Cells

Heterogeneous and ¹³C-labelled colonies were grown on SM medium with 1% ¹³C glucose and 200 µM lysine for three days. Colonies were then resuspended in 1.1 ml of SM with 0.2% ¹³C glucose, and 500 µl was added to a similar volume of amphotericin B solution followed by mixing and incubation for one h as described above. Cells were collected by centrifugation and resuspended in PBS. Before FACS, cells were sonicated for 20 s at 50 W (JSP Ultrasonic Cleaner model US21) to increase singlet efficiency. Cells were then stained with 8 µg ml^–1 propidium iodine to identify live and dead cells, before FACS analysis. Live and dead cells were sorted on a BD Aria Fusion with BD FACSDiva (v.8.0.1) software (BD Biosciences) using a 488 nm excitation laser. The gating strategy is illustrated in Extended Data Fig. 7a. Sorted cells were collected by filtration through a 0.45 µm polyvinylidene difluoride membrane (Agilent, no. 200959–100) and washed from the filter with 200 µl of proteomics lysis buffer, followed by sample processing and targeted PRM measurement as described above.