Applying Proteomic Method To Invest The Impact Of Peptide P2 On The FGF Signal Transduction Pathway

The fibroblast growth factor (FGF) family, composed of at least 25 members, was characterized as a polypeptide with mitogenic properties. FGF acts several bioactivities by binding its high-affinity receptors, FGFR, with the help of heparin or heparan sulfate proteoglycans (HSPG). FGF/FGFR signal transduction pathway is a phosphorylation cascade amplification course, which plays an critical role in embryonic development, tissue repair, wound healing and angiogenesis. Many diseases are correlated with this signal transduction pathway, such as cancer , skeletal dysplasias and olfactory syndromes.The peptide P₂ (VYMSPF) that was selected from the phage display peptide library is an antagonist of aFGF. The computer predicted that P₂ can competitively bind to the hydrophobic surface of the receptor. In order to invest the impact of P₂ on FGF signal transduction pathway, we synthesized it with the solid phase peptide synthesis method. Then we cultured NIH3T3 cell stimulated with different conditions. No.1 without any stimulation, No.2 stimulated with aFGF and heparin, No.3 stimulated with aFGF, heparin and P₂. After that, we separate the whole protein and phosphoprotein with two dimensional electrophoresis, handle the images with ImageMaster 2D software and identify them with ESI-MS/MS.As the purity of the proteome plays an important role in the result of the 2DE, we optimized the sample preparation proceeding. Besides the regular cell lysising method, we applied acetone precipitation to desalt the sample. Finally, we got clear images by eliminating the longitudinal striation and transverse striation. After separating the protein with pH3-10 IPG stripe (linear), we found that most of the protein was acid, so we separated it with the IPG stripe(linear) of pH4-7 to get a better resolution. All spots were repeated three times before analyzed by mass spectrometry.We identified five spots with two fold's difference between sample No.2 and 3. Four of them are down-regulated, spot 1 is Actin, cytoplasmic 1;spot 2 is Alpha-enolase;spot 3 is Eukaryotic translation initiation factor 6;spot 5 is Alpha-enolase, spot 4 is up-regulated which is Triosephosphate isomerase.We applied affinity chromatography to enrich and purify the phosphoprotein of the cells. As the concentration is too low, and the salt was too hard to elimate, the method is still under groping.