13c6 lysine

NOZ cells were grown in a ¹³C₆-lysine/¹³C₆-arginine-containing (heavy) medium, while NOZ GemR cells were grown in a normal (light) medium. The experiment was carried out in a biological duplicate. DMEM with and without lysine and arginine, fetal bovine serum (FBS), L-glutamine, and antibiotics were purchased from Thermo Fisher Scientific. SILAC amino acids, ¹³C₆-lysine, and ¹³C₆-arginine were acquired from Cambridge Isotope Laboratories (Andover, MA, USA). Peptides were prepared using an in-solution tryptic digestion protocol with modifications [54 (link),55 (link)]. The eluted peptides were lyophilized and subjected to phosphopeptide enrichment by immunoaffinity purification (IAP), as previously described [54 (link),55 (link)]. Peptides were eluted twice from beads by incubating the beads with 0.1% TFA at room temperature. Protocol details are shown in the Supplementary Materials: Supplementary Methods/SILAC protocol.

Extraction buffer was prepared by adding 2:2:1 methanol:acetonitrile:water to internal standards at 1 μg/mL each (D4-Citric Acid, ¹³C5-Glutamine, ¹³C5-Glutamic Acid, ¹³C6-Lysine, ¹³C5-Methionine, ¹³C3-Serine, D4-Succinic Acid, ¹³C11-Tryptophan, and D8-Valine; Cambridge Isotope Laboratories). Plasma volume was included in the calculations for the water portion of the buffer. A total of 720 µL of extraction buffer was then added to 40 μL of each plasma sample. Samples were placed on a rotating platform at –20 °C for 1 h and centrifuged at 4 °C for 10 min at 21,000× g. A total of 300 µL of the metabolite extracts was transferred into fresh microcentrifuge tubes. An equal volume of each extract was pooled to serve as a quality control (QC) sample, which was analyzed at the beginning, end, and after every tenth sample throughout the instrument run. Extraction buffer alone was analyzed as a processing blank sample to account for carryover or background contamination from the sample extraction process. Metabolite extracts, the QC sample, and the processing blank were evaporated to dryness using a speed-vacuum. All samples were reconstituted in 30 μL of acetonitrile/water (1:1, v/v), vortexed for 10 min, and incubated at –20 °C for 18 h. Samples were then centrifuged at 4 °C for 2 min at 21,000× g and the supernatant was transferred into autosampler vials for analysis.