Analysis Of Phosphorylated Proteomics To The MDA-MB-468 Nucleus In Response To EGF Stimulation

Epidermal growth factor receptor is an important transmembrane receptor with signal-transducing tyrosine kinase activity. Emerging evidences suggest activated EGFR undergoes nuclear translocation upon EGF stimulation and subsequently regulates gene expression and signaling transduction. However, there is still a lack of understanding about extensive nucleus phosphorylation response after EGFR nuclear translocation. Here, we performed preliminary quantitative phosphoproteomics analysis in MDA-MB-468 nucleus using iTRAQ technology.Firstly, we tested whether EGFR can translocate into nucleus on different time points. We observed obviously EGFR translocation after EGF treatment for 15min. Then nucleus fragment were prepared by differential centrifugation. After extraction and digestion of nuclear proteins, we labeled the peptides with iTRAQ reagents. Then we identified peptides and proteins by using highly sensitive TiO2 based phosphopeptide enrichment compatible with TripleTOF 5600 mass spectrometry analysis. Further, Scaffold and scaffold the PTM software were performed on the MS signal peptide search and protein quantitation.Scaffold qualitative analysis showed that 275 proteins in the nucleus of MDA-MB-468 were identified, of which 242 were phosphorylated proteins, corresponding to 336 phosphorylated peptides and 405 phosphorylation sites. Scaffold quantitative analysis showed that 87 phosphorylated proteins exhibited significant abundance changes (1.5-fold difference) after EGF stimulation, including 114 phosphopeptides and 137 phosphorylation sites. Organelle classification showed that 63 of them locate in the nucleus. Of which 42 subject to the activation of cytoplasmic EGFR signaling because their upstream kinases are MAPK,GSK,PKC; However, two proteins:KHDRBS1and TNKS1BP1 are possible indirect nuclear EGFR substrate in response to EGF stimulation. Other 19 proteins with unknown upstream kinase may be nuclear EGFR downstream signal molecules in response to EGF stimulation. In-depth study will provide important clues to explore the molecular mechanism of nuclear translocation of EGFR.