Boeri Erba Elisabetta, Matthiesen Rune, Bunkenborg Jakob, Schulze Waltraud X, Di Stefano Paola, Cabodi Sara, Tarone Guido, Defilippi Paola, Jensen Ole N
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
J Proteome Res. 2007 Jul;6(7):2768-85. doi: 10.1021/pr060675m. Epub 2007 May 25.
Using stable isotope labeling and mass spectrometry, we performed a sensitive, quantitative analysis of multiple phosphorylation sites of the epidermal growth factor (EGF) receptor. Phosphopeptide detection efficiency was significantly improved by using the tyrosine phosphatase inhibitor sodium pervanadate to boost the abundance of phosphorylation of the EGF receptor. Nine phosphorylation sites (pT669, pS967, pS1002, pY845, pY974, pY1045, pY1086, pY1148, and pY1173) of EGF receptor were quantified from EGF-stimulated cells in suspension and adherent conditions. Our data sets revealed that EGF stimulation of adherent cells induced higher levels of tyrosine phosphorylation relative to EGF stimulation of suspended cells. In contrast, EGF stimulation of adherent cells induced lower levels of serine and threonine phosphorylation relative to EGF stimulation of suspended cells. These findings are consistent with the hypothesis that cellular adhesion modulates phosphorylation of plasma membrane receptor tyrosine kinases relevant for EGF-induced signal transduction processes. Furthermore, our results suggest that strong phosphatase inhibitors should be used to generate reference datasets in comparative phosphoproteomics experiments.
我们使用稳定同位素标记和质谱技术，对表皮生长因子（EGF）受体的多个磷酸化位点进行了灵敏的定量分析。通过使用酪氨酸磷酸酶抑制剂过氧钒酸钠提高EGF受体的磷酸化丰度，显著提高了磷酸肽的检测效率。从悬浮和贴壁条件下的EGF刺激细胞中定量分析了EGF受体的9个磷酸化位点（pT669、pS967、pS1002、pY845、pY974、pY1045、pY1086、pY1148和pY1173）。我们的数据集显示，相对于悬浮细胞的EGF刺激，贴壁细胞的EGF刺激诱导了更高水平的酪氨酸磷酸化。相反，相对于悬浮细胞的EGF刺激，贴壁细胞的EGF刺激诱导了更低水平的丝氨酸和苏氨酸磷酸化。这些发现与细胞黏附调节与EGF诱导信号转导过程相关的质膜受体酪氨酸激酶磷酸化的假设一致。此外，我们的结果表明，在比较磷酸蛋白质组学实验中应使用强磷酸酶抑制剂来生成参考数据集。
