ABSTRACT: To ensure that the pool of precursor amino acids utilized for protein synthesis is completely labeled prior to the first labeling time-point, we conducted an isotopomer analysis. After adaption to media, cells were plated at a density of 500,000 cells per 10 cm plate. 8 days after plating, the confluent quiescent cultures were switched to 15N labeling medium. Cells were collected after 0h, 6h, 1d, 2d, 4d of labeling, washed with PBS and cell pellets were frozen prior to further analysis. 15N labeling medium was made from “Cell free” Amino Acid Mix (20AA, U-15N, 96-98%)(Cambridge Isotope Laboratories) and supplemented with 15% dialyzed fetal bovine serum (Thermo Scientific), 100U/mL penicillin, 100U/mL streptomycin. 1g is used for making 702mL labeling medium. The final amino acids concentrations in 15N labeling medium are as following: Alanine: 0.1140 g/L, Arginine: 0.0499 g/L, Asparagine: 0.0940 g/L, Aspartic acid: 0.1353 g/L, Cystine: 0.01140 g/L, Glutamic acid: 0.1852 g/L, Glutamine: 0.0869 g/L, Glycine: 0.0698 g/L, Histidine: 0.0157 g/L, Isoleucine: 0.0698 g/L, Leucine: 0.1211 g/L, Lysine: 0.0527 g/L, Methionine: 0.0228 g/L, Phenylalanine: 0.0541 g/L, Proline: 0.0299 g/L, Serine: 0.0541 g/L, Threonine: 0.0741 g/L, Tryptophan: 0.0456 g/L, Tyrosine: 0.0613g/L, Valine: 0.0798 g/L. LC-MS/MS and quantitative analysis of isotopic envelopes and 15N incorporation of peptides was conducted as described in (Zhang et al., 2014). The data indicate that when exposed to fully 15N-labeled media (where all 20 natural amino acids are isotopically labeled), the fraction of proteins that are synthesized within the first day are almost fully labeled. Thus, the extent of fractional labeling is almost exclusively influenced by the kinetics of protein turnover rather than amino acid uptake and recycling within the cell. Table of files Cells Time points Labeling Fractionation Raw Data Filenames Wildtype 0d, 1d,2d,4d,7d13C 6Lys, 6Arg& 15N No 0d.raw 1d.raw 2d.raw 4d.raw 7d.raw
Tian Zhang 
