13c615n2 lysine

These procedures have been described in detail [14 (link)]. Briefly, arginine and lysine auxotrophic Escherichia coli AT713 bacteria were cultured in M9 basal medium supplemented with arginine (100 μg/ml), cysteine (100 μg/ml) and lysine (100 μg/ml either ¹²C₆,¹⁴N₂-lysine) or ¹³C₆,¹⁵N₂-lysine) (Cambridge Isotope Laboratories, Andover, MA)) (M9 with amino acid supplementation) in an incubator shaker (3°C, 200 rpm until OD₆₀₀ reached 1.5), pelleted by centrifugation, and killed with 100% ethanol.
Age-synchronized animals were cultured to day 3 on peptone-free NGM plates seeded with regular or heavy lysine-labeled AT713 bacteria along with 25 mg/l 5′-FUDR (5-fluoro-2′deoxyuridine, Geel, Belgium) starting from day 0. Then bacteria were separated from nematodes by an H₂O wash and nematodes were pelleted by centrifugation. Equal weights of heavy lysine-labeled and unlabeled WT worms were suspended in 100 mM ammonium bicarbonate containing 4% perfluorooctanoic acid (w/v), and proteins were extracted by ultrasonication (4.5 kHz three times for 9 s with a 3-min pause on ice between pulses) using a Virsonic 100 ultrasonic cell disrupter (SP Scientific, Warminster, PA). Extracted proteins were reduced with dithiothreitol and S-alkylated with iodoacetamide, and then digested by Lys-C as described previously [14 (link)].

HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) with 10% fetal bovine serum (Hyclone, South Logan, Utah) and penicillin-streptomycin (100U/mL and 100μg/mL, Invitrogen), at 37°C in a humidified atmosphere with 5% CO₂. For SILAC experiments, HeLa cells were grown in arginine- and lysine-free DMEM with 10% dialyzed fetal bovine serum (Hyclone) supplemented with either 100mg/L ¹³C₆¹⁵N₂-lysine and 100mg/L ¹³C₆¹⁵N₄-arginine (Cambridge Isotope Laboratories, Inc., Andover, MA) (“heavy”) or identical concentrations of isotopically-normal lysine and arginine (“light”) for at least six cell doublings.

N^α-Boc-L-Lysine and N^α-acetyl-L-Lysine used as surrogates for protein lysyl residues were purchased from Chem-Impex International, Inc. (Wood Dale, Illinois). D₃-2-aminoadipic acid was purchased from C/D/N isotopes Inc. (Pointe-Claire, Quebec). ¹³C₆,¹⁵N₂-Lysine was purchased from Cambridge isotope laboratories, Inc. (Andover, Massachusetts). Sodium hypochlorite (NaOCl), H₂O₂, ammonium hydroxide (NH₄OH), trifluoroacetic acid and organic solvents were obtained from Fisher Scientific Co. Pronase was purchased from Roche life science (Indianapolis, Indiana). SAX SPE cartridges were obtained from Jordi lab (Mansfield, Massachusetts). Axis-Shield Polymorphprep was purchased from Cosmo Bio USA (Carlsbad, California). All other materials were obtained from Sigma-Aldrich, unless otherwise indicated.