PROTEOMIC ANALYSIS OF SYNAPTIC MOLECULAR STRUCTURES AND FUNCTIONAL REGULATION BY PROTEIN PHOSPHORYLATION

本研究项目有两个目的。(1)To阐明蛋白磷酸化对突触前神经递质释放的调节机制。(2)To分析突触分子结构，包括信号转导通路。对于前者，应采用超灵敏的质谱分析来分析突触囊泡和SNARE复合物中蛋白质的磷酸化。对于后者，同样的质谱方法被应用于分析在生长锥中发现的组分蛋白以及突触后密度。同时，对后一项研究中发现的这些蛋白质的蛋白磷酸化进行分析。为了实现这些研究，我们建立了一个以五个质谱仪为中心的蛋白质组学设施。该设施还包括各种自动机器人，例如凝胶拾取机器人和凝胶内消化机器人。我们建立了由各种机器人组成的高通量分析系统。将三级四极杆质谱仪与毛细管高效液相色谱仪联用，采用前体扫描法对磷酸肽进行分析。在这些研究中，我们在脑特异性微管相关蛋白MAP1B中鉴定了50多个磷酸化位点，并鉴定了几个突触融合蛋白结合蛋白。

The present research project has two aims. (1)To elucidate the regulatory mechanisms of presynaptic neurotransmitter release by protein phosphorylation. (2)To analyze the synaptic molecular structure including sinal transduction pathways. For the former, ultra-sensitive mass spectrometric analysis should be applied to analyze the protein phosphorylation of proteins in the synaptic vesicles and in the SNARE complex. For the latter, the same mass spectrometric method is applied to analyze the component proteins found in the growth cones as well as the postsynaptic densities. At the same time, the protein phosphorylation of these proteins found in the latter study should be analyzed. To achieve these studies, we have established a proteomics facility centered on five mass spectrometers. This facility includes also various automatic robots such as gel picking robot and in-gel digestion robot. We have established a high-throughput analysis system consisting of various robots. A triple-stage quadrupole mass spectrometer was combined with a capillary HPLC to conduct phosphopeptide analysis based on precursor scanning method. During these research we could identify more than 50 phosphorylation sites in a brain-specific microtubule-associated protein MAP1B.We have also identified several syntaxin-binding proteins.