MassIVE MSV000081638

Partial Public PXD008026

MudPIT analyses of whole cell extracts from Y1 murine adrenocortical carcinoma cell line stimulated with serum or FGF2

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Description

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. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Fibroblast growth factor 2 ; fosB ; junB ; src ; replication ; mice tumor cell

Contact

Principal Investigators:
(in alphabetical order) 		Laurence Florens, The Stowers Institute for Medical Research, USA
Submitting User: laflorens

Publications

Vitorino FNL, Montoni F, Moreno JN, de Souza BF, Lopes MC, Cordeiro B, Fonseca CS, Gilmore JM, Sardiu MI, Reis MS, Florens LA, Washburn MP, Armelin HA, da Cunha JPC.
FGF2 Antiproliferative Stimulation Induces Proteomic Dynamic Changes and High Expression of FOSB and JUNB in K-Ras-Driven Mouse Tumor Cells.
Proteomics. 2018 Sep;18(17):e1800203. Epub 2018 Aug 14.

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