Systems-wide analysis of a phosphatase knock-down by quantitative proteomics and phosphoproteomics.
Systems-wide analysis of a phosphatase knock-down by quantitative proteomics and phosphoproteomics.
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DOI：
10.1074/mcp.m800559-mcp200
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发表时间：
2009-08
期刊：
影响因子：
--
通讯作者：
Mann M
中科院分区：
文献类型：
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作者：
Hilger M;Bonaldi T;Gnad F;Mann M
后生动物的信号转导几乎调节着生物学功能的所有方面，异常信号转导参与了许多疾病。基于磷酸化的信号网络中的扰动通常使用磷酸特异性抗体以假设驱动的方法进行研究。在这里，我们应用定量，高分辨率质谱法来确定系统响应一个信号成分的耗尽。果蝇细胞代谢标记使用稳定同位素标记的氨基酸在细胞培养物（SILAC）和磷酸酶Ptp61 F，直系同源的哺乳动物PTB1B，糖尿病的药物靶点，被敲低的RNAi。我们总共在果蝇Schneider细胞的磷酸化蛋白质组中检测到超过10，000个磷酸化位点，并使用这些数据训练磷酸化位点预测器。磷酸酶敲低后基于SILAC的定量显示，除了磷酸酶外，蛋白质组受到的影响最小，而6，478个高置信度磷酸化位点中有288个发生了显著变化。在磷酸酪氨酸水平的反应包括已经描述的Ptp61F底物Stat92E和阿比特龙。我们的分析强调了连接Ptp61F的细胞骨架调节通过GTdR调节蛋白质和黏着斑成分。
Signal transduction in metazoans regulates almost all aspects of biological function, and aberrant signaling is involved in many diseases. Perturbations in phosphorylation-based signaling networks are typically studied in a hypothesis-driven approach, using phospho-specific antibodies. Here we apply quantitative, high-resolution mass spectrometry to determine the systems response to the depletion of one signaling component. Drosophila cells were metabolically labeled using stable isotope labeling by amino acids in cell culture (SILAC) and the phosphatase Ptp61F, the ortholog of mammalian PTB1B, a drug target for diabetes, was knocked down by RNAi. In total we detected more than 10,000 phosphorylation sites in the phosphoproteome of Drosophila Schneider cells and trained a phosphorylation site predictor with this data. SILAC-based quantitation after phosphatase knock-down showed that apart from the phosphatase, the proteome was minimally affected whereas 288 of 6,478 high-confidence phosphorylation sites changed significantly. Responses at the phosphotyrosine level included the already described Ptp61F substrates Stat92E and Abi. Our analysis highlights a connection of Ptp61F to cytoskeletal regulation through GTPase regulating proteins and focal adhesion components.