L methionine

PC(12:0/13:0), PE(12:0/13:0), SM(d18:1/12:0) and Cer(d18:1/12:0) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Stable isotope labeled references were purchased from Cambridge Isotope Laboratories (Andover, MA, USA), including: Glycine (2-¹³C, ¹⁵N), L-Alanine (2,3,3,3-D₄), L-Valine (D₈), L-Leucine (5,5,5-D₃), L-Methionine (methyl-D₃), L-Phenylalanine (ring-¹³C₆), L-Tyrosine (ring-¹³C₆), L-Aspartic acid (2,3,3-D₃), DL-Glutamic acid (2,4,4-D₃), L-Ornithine: HCl (5,5-D₂), L-Citrulline (5,5-D₂) and L-Arginine (5-¹³C, 4,4,5,5-D₄). Deuterium-labeled carnitine and acylcarnitines (NSK-B set) were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA), including ²H₃-Acetylcarnitine (C₂), ²H₃-Propionylcarnitine (C₃), ²H₃-Butyrylcarnitine (C₄), ²H₉-Isovalerylcarnitine (C₅), ²H₃-Octanoylcarnitine (C₈), ²H₉-Myristoylcarnitine (C₁₄), and ²H₃-Palmitoylcarnitine (C₁₆). HPLC-grade ammonium acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA), and HPLC-grade formic acid and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All solutions were prepared by LC-MS ultra-pure water.

C57bl6/j mice were obtained from Jackson Laboratories (Stock #000664). Labeled internal standards citric acid (2,2,4,4-D4, 98%; #DLM-3487), succinic acid (2,2,3,3-D4, 98%; #DLM-584), l-valine (2,3,4,4,4,5,5,5-D8; 98%; #DLM-311), l-glutamic acid (¹³C5, 99%; #CLM-1800), l-glutamine (¹³C5, 99%, #CLM-1822), l-lysine (¹³C6, 99%; #CLM-2247), l-methionine (¹³C5, 99%; #CLM-893), and l-tryptophan (¹³C11, 99%; #CLM-4290) and tracer glucose (¹³C6, 99%, #CLM-1396) were obtained from Cambridge Isotope Laboratories, Inc. AAV8-TBG-NULL (#105536-AAV8) and AAV8-TBG-Cre (#107787-AAV8) were purchased from Addgene. Pre-filled bead mill tubes containing 1.4 mm ceramic beads (#15-340-153) were purchased from FisherScientific. Methoxaymine hydrochloride (#226904, MOX) and N-methyl-N-(trimethylsilyl) trifluoroacetamide (#694709, MSTFA) were purchased from Sigma Aldrich. Protease inhibitor cocktail (#786-437) was purchased from G Biosciences. Bio-Rad protein assay dye regent (#5000006) and 0.45 μm nitrocellulose membrane (#1620115) were purchased from Bio-Rad.

The heavy methyl-SILAC experiment was conducted based on the principles and procedure described in earlier researches (68 (link), 112 (link), 113 ). The SILAC labeling media were prepared by reconstituting commercially available DMEM high-glucose media depleted for glutamine, methionine, and cysteine (21013024; DMEM high glucose, no glutamine, no methionine, no cysteine cell culture media, Thermo Fisher scientific). For reconstitution, we added 4 mM glutamine (25030-081, Thermo Fisher scientific), 200 μM cysteine (1001527621, L-cysteine dihydrochloride, Sigma-Aldrich) and either 200 μM normal methionine (1001818815; L-Methionine, Sigma-Aldrich) for the light medium or 200 μM heavy methionine (CDLM9289025, L-Methionine, Methyl-13C 99%, Methyl-D3 98%, Cambridge Isotope Laboratories) for the heavy medium. After reconstitution, we added 10% dialyzed FBS (F0392, Sigma-Aldrich) and a combination of 100 U/ml penicillin and 100 μg/ml streptomycin (15140-122, Penicillin-Streptomycin, Thermo Fisher scientific). HeLa cells were cultured in either light or heavy medium for at least six generations. The heavy-labeled cells were treated with 30 mM cLEU for 6 h before harvest. The ribosome was purified as follows: