Quantitative proteomics analysis of insulin and insulin receptor agonist S597 signaling in rat hepatoma cell line

These data files are collected from Stable Isotope Labeling with Amino acids in Cell culture (SILAC) mass spectrometry (MS)-based proteomics and phospho-proteomics experiments. The rat hepatoma cell line, H4IIE, were exposed to SILAC for at least 14 days in Minimum Essential Medium (MEM) for SILAC added 1% Penicillin/Streptomycin, 10% dialyzed fetal bovine serum for use in SILAC, 1% MEM non-essential amino acids solution, sodium pyruvate, and GlutaMAX. The three SILAC conditions comprised the following three conditions: Light (Lys0,Arg0), medium (Lys4,Arg6), and heavy (Lys8,Arg10). For the phospho-proteome analysis, SILAC H4IIE cells were starved overnight and then stimulated with vehicle (light), insulin (medium), and the partial insulin receptor agonist S597 (heavy) for 5, 15, or 30 minutes. After stimulation, the cells were washed in Dulbecco's Phosphate-Buffered Saline (DPBS) and lysed in Guanidine-HCl added PhosSTOP. Protein concentrations were measured by BCA. Two independent sets of biological replicates were collected. For the first biological replicate, 2 mg of protein from each treatment condition was mixed in a 1:1:1 ratio to give a final protein content of 6 mg and for the second 4 mg from each treatment condition was mixed. For proteome analysis, SILAC H4IIE cells were treated with vehicle (light), insulin (medium), and S597 (heavy) for 24 or 48 hours in regular SILAC medium. For the 48-hour samples, the media was changed after 24 hours of stimulation. Following treatment, the cells were washed in DPBS and lysed with RapiGest, and protein concentrations were measured by BCA. To prepare the proteins for digestion, 200 µg of proteins from each treatment condition was mixed in a 1:1:1 ratio for 3 independent biological replicates.