SPECIFIC PROTEOME OF MAMMALIAN CORTEX INHIBITORY & EXCITATORY SYNAPSES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 四年前，我们与Nat Heintz(洛克菲勒大学)合作，开始了对浦肯野细胞兴奋性突触中存在的蛋白质补体的研究。我们使用细菌人工染色体(BAC)修饰策略来靶向GFP融合的网格2到浦肯野细胞的兴奋性突触的体内特异性表达。我们对小鼠小脑进行了解剖，并纯化了带有GFP-GRID2的突触。尽管具有挑战性，但我们的方法被证明是成功的，因为我们分离了突触并分析了低水平的Femtomol蛋白质。在过去的一年里，我们继续我们的质谱学分析，鉴定了约70个突触蛋白，证实了已知的兴奋性蛋白，不存在抑制性蛋白，并鉴定了兴奋性突触的新特征。我们已经发表了一份描述这项工作的手稿(F.Slimi，I.Cristea，E.Heller，B.T.Chait，N.Heintz：《单一中枢神经系统突触类型的蛋白质组学研究：平行纤维/浦肯野细胞突触》PLoS Biology，2009年4月14日；7(4)：E83)。 使用与上述方法类似的方法，我们目前正在通过从大脑皮层特定细胞群中分离GABA受体来研究抑制性突触的蛋白质组成。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Four years ago, in collaboration with Nat Heintz (Rockefeller University), we initiated the study of the protein complement present at excitatory synapses in Purkinje cells. We used the Bacterial Artificial Chromosome (BAC) modification strategy to target the specific in vivo expression of GFP-fused GRID 2 to Purkinje cell's excitatory synapses. We performed dissections of mouse cerebella, and purified synapses bearing GFP-GRID2. Although challenging, our approach proved successful, as we isolated synapses and analyzed low-femtomol levels of proteins. During this last year, we continued our mass spectrometric analyses and identified ~70 synaptic proteins, confirming known excitatory proteins, the absence of inhibitory proteins, and identifying novel signatures of excitatory synapses. We have published a manuscript describing this work (F. Selimi, I. Cristea, E. Heller, B.T. Chait, N. Heintz "Proteomic studies of a single CNS synapse type: the parallel fiber/Purkinje cell synapse" PLoS Biology, 2009 Apr 14;7(4):e83). Using a similar approach to the one described above, we hare currently studying the protein composition of inhibitory synapses by isolating GABA receptors from specific cell populations in the Cortex.