Fibroblast growth factor 2 (FGF2) is well known as a promoter of cell proliferation, however, in some cellular contexts, it can induce cell cycle arrest or cell death. In Y1 murine adrenocortical carcinoma cell line, FGF2 induces G0/G1 transition but delays S-phase progression and permanently block cells in G2/M. To gain insights into the molecular mechanisms of this antiproliferative effect, we investigated the early proteomics alterations induced by this growth factor. We compared mass spectrometry-based quantitative proteomics analyses of Y1 cells stimulated, after 3 and 5 hours, with fetal bovine serum (FBS) or FGF2. Total protein extracts from Y1 cells stimulated with FBS or FBS plus FGF2 (10 ng/ml) for 0 h, 3 h, and 5 h were obtained in 3 biological replicates. One hundred and fifty micrograms of proteins were TCA precipitated and dissolved in 100 mM Tris-HCL pH 8.5, 8 M urea. The sample was reduced with 5 mM TCEP, incubated at room temperature for 30 min, and carboxymethylated by the addition of 10 mM of iodoacetamide. Proteins were digested with endoproteinase Lys-C for 4 h and then digested with Trypsin overnight at 37C. The peptides were loaded onto a 100 um three-phase capillary column packed with 8 cm of 5 um C18 reverse phase (Aqua; Phenomenex), followed by 3 cm of 5 um SCX resin (Partisphere SCX; Whatman) and an additional 1.5 cm of reverse phase. Columns loaded with the peptides were placed in-line with an Agilent 1100 quaternary HPLC connected with a LTQ-XL (Thermo) mass spectrometer equipped with a nanospray ionization source. Twelve automatic steps were used in the MudPIT run with increasing concentrations of ammonium acetate. Each full MS scan (400 m/z to 1600 m/z) was followed by 5 MS/MS events fragmented by CID.