Towards a Dendritic Proteome

描述（由申请人提供）：在神经元中，越来越多的证据表明，局部树突蛋白合成用于允许单个突触动态响应伴随突触连接的建立、维持和可塑性的环境变化。此外，现在已经很好地确定，翻译机制的基本组成部分以及mRNA存在于突触处或附近，树突可以合成新的蛋白质。尽管如此，到目前为止，只鉴定了有限数量的局部翻译蛋白质，主要是通过基于候选人的方法，其灵感来自特定实验室对单个神经炎过程中mRNA产生的蛋白质“x”或“y”的兴趣以及随后的PCR实验。然而，我们相信，专注于翻译的蛋白质是必不可少的突触和树突如何能够安装在环境中的局部变化的反应的准确图片。目前可用的评估细胞或细胞区室的蛋白质组成变化的方法依赖于两个蛋白质组彼此之间的比较。只有当蛋白质以足够的量存在以被检测时，并且当比较的蛋白质组之间的蛋白质表达水平存在很大差异时，这些差异方法才是成功的。在这里，我们描述了一种新的高通量蛋白质组分析技术的发展，以分离和识别神经元树突中的翻译产物，并确定在基础和刺激条件下树突的定量翻译能力。具体来说，我们提出了一个程序，使用人工氨基酸标记新合成的树突状蛋白在原位，从而规避传统方法的问题。获得的蛋白质组数据集将有助于我们理解局部翻译的逻辑。这项技术可以很容易地应用于许多蛋白质组分析问题，无论组织类型或细胞背景。树突状蛋白的变化，与可塑性，包括成瘾相关的突触修饰的基础上，可以阐明使用这种技术。

DESCRIPTION (provided by applicant): In neurons, there is increasing evidence that local dendritic protein synthesis is used to allow individual synapses to respond dynamically to the environmental changes that accompany the establishment, maintenance and plasticity of synaptic connections. Furthermore, it is now well established that the essential components of the translational machinery as well as mRNAs are present at or near synapses and that dendrites can synthesize new proteins. Despite this, only a limited number of locally translated proteins have been identified so far, mainly through candidate-based approaches inspired by a particular laboratory's interest in protein "x" or "y" ¿r by mRNA generation from single neuritic process with subsequent PCR experiments. However, we believe that a focus on translated proteins is essential for an accurate picture of how the synapse and the dendrite can mount a response to local changes in the environment. Current available methods to evaluate changes in the protein composition of a cell or of a cellular compartment rely on the comparison of two proteomes with one another. These differential approaches are successful only when proteins are present in sufficient quantity to be detected and when there are large differences in protein expression levels between the compared proteomes. Here we describe the development of a new high-throughput proteomic profiling technique to isolate and identify the translation products in neuronal dendrites and to determine the quantitative translational capability of dendrites under both basal and stimulated conditions. Specifically, we propose a procedure that uses artificial amino acids for the labeling of newly synthesized dendritic proteins in situ, thereby circumventing problems of traditional approaches. The obtained proteomic datasets will help us to understand the logic of local translation. This technology can be easily applied to many proteomic profiling questions, regardless of tissue type or cellular context. The changes in dendritic proteins that underly the synaptic modifications associated with plasticity, including addiction, can be elucidated using this technique.