Soskic V, Görlach M, Poznanovic S, Boehmer F D, Godovac-Zimmermann J
Institute for Molecular Biotechnology, Jena, Germany.
Biochemistry. 1999 Feb 9;38(6):1757-64. doi: 10.1021/bi982093r.
We report efficient methods for using functional proteomics to study signal transduction pathways in mouse fibroblasts following stimulation with PDGF. After stimulation, complete cellular proteins were separated using two-dimensional electrophoresis and phosphorylated proteins were detected with anti-phosphotyrosine and anti-phosphoserine antibodies. About 260 and 300 phosphorylated proteins were detected with the anti-phosphotyrosine and anti-phosphoserine antibodies, respectively, at least 100 of which showed prominent changes in phosphorylation as a function of time after stimulation. Proteins showing major time-dependent changes in phosphorylation were subjected to in-gel digestion with trypsin and identified by mass spectroscopy using MALDI-TOF mass fingerprinting and ESI peptide sequencing. We have observed phosphorylated proteins known to be part of the PDGF signal transduction pathway such as ERK 1, serine/threonine protein kinase akt and protein tyrosine phosphatase syp, proteins such as proto-oncogene tyrosine kinase fgr previously known to participate in other signal transduction pathways, and some proteins such as plexin-like protein with no previously known function in signal transduction. Information about the phosphorylation site was obtained for proto-oncogene tyrosine kinase fgr and for cardiac alpha-actin. The methods used here have proven to be suitable for the identification of time-dependent changes in large numbers of proteins involved in signal transduction pathways.
我们报告了利用功能蛋白质组学研究血小板衍生生长因子（PDGF）刺激后小鼠成纤维细胞信号转导途径的有效方法。刺激后，使用二维电泳分离完整细胞蛋白，并用抗磷酸酪氨酸和抗磷酸丝氨酸抗体检测磷酸化蛋白。分别用抗磷酸酪氨酸和抗磷酸丝氨酸抗体检测到约260种和300种磷酸化蛋白，其中至少100种在刺激后的磷酸化水平随时间呈现显著变化。对磷酸化水平呈现主要时间依赖性变化的蛋白质进行胰蛋白酶胶内消化，并通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）质谱指纹图谱和电喷雾电离（ESI）肽测序进行质谱鉴定。我们观察到已知为PDGF信号转导途径一部分的磷酸化蛋白，如细胞外信号调节激酶1（ERK 1）、丝氨酸/苏氨酸蛋白激酶Akt和蛋白酪氨酸磷酸酶syp，先前已知参与其他信号转导途径的原癌基因酪氨酸激酶fgr等蛋白质，以及一些在信号转导中以前未知功能的蛋白，如类丛状蛋白。获得了原癌基因酪氨酸激酶fgr和心肌α-肌动蛋白的磷酸化位点信息。这里使用的方法已被证明适用于鉴定信号转导途径中大量蛋白质的时间依赖性变化。
