13c6 15n4 arginine

SILAC is a simple and straightforward approach for in vivo incorporation of a label into proteins for mass spectrometry (MS)-based quantitative proteomics. Cells were cultured in conventional RPMI 1640 medium containing ¹²C¹⁴N-Lysine and ¹²C¹⁴N-Arginine (“light”) overnight. The cells were then transferred into RMPI 1640 medium containing ¹³C₆¹⁵N₂-Lysine and ¹³C₆¹⁵N₄-Arginine (“heavy”) (Sigma) with or without 50 nM of SS-31. After 24 h, the cells were collected and lysed with lysis buffer. Total FXN was enriched by immunoprecipitation with FXN antibody and assayed by LC-MS/MS using machine Q Exactive (Thermo Fisher Scientific). Newly synthesised FXN containing the “heavy” isotopes is distinguishable from the “old” FXN containing the “light” isotopes.

HeLa (ATCC: CCL-2) cells were tested for mycoplasma contamination and grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin. SILAC media was supplemented with arginine and lysine (SILAC Light) or with heavy isotope-labeled arginine (¹³C_6,¹⁵N₄-arginine, Sigma) and lysine (¹³C_6,¹⁵N₂-lysine, Cambridge Isotope Laboratories) (SILAC heavy) in media containing dialyzed serum (Sigma). Cells were cultured at 37 °C in a humidified incubator at 5% CO₂. At a confluency of ~90%, cells were washed twice with PBS and lysed in ice-cold modified RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1x mini complete protease inhibitor cocktail (Roche), 10 mM nicotinamide, and 5 μM trichostatin A). Lysates were mixed with 1/10 volume of 5 M NaCl to release chromatin-bound proteins and incubated for 15 min on ice. Subsequently, lysates were homogenized by sonication (6 × 10 sec, 15 W), cleared by centrifugation (20,000 × g, 15 min, 4 °C), and the supernatant precipitated by addition of four volumes of −20 °C acetone. Precipitates were re-dissolved in 8 M guanidine HCl, 50 mM Hepes pH8.5 and protein concentration was determined by Quick-start Bradford assay (Bio-Rad).