Towards a Dendritic Proteome

描述(申请人提供)：在神经元中，越来越多的证据表明，局部树突蛋白合成被用来允许单个突触对伴随着突触连接的建立、维持和可塑性的环境变化做出动态反应。此外，现在已经很好地证实，翻译机制的基本组件以及mRNAs存在于突触或突触附近，树突可以合成新的蛋白质。尽管如此，到目前为止，只有有限数量的本地翻译蛋白质被鉴定出来，主要是通过基于候选的方法，受到特定实验室对蛋白质“x”或“y”？r的兴趣的启发，这些蛋白质是通过随后的PCR实验从单个神经细胞过程中产生的mRNA而产生的。然而，我们认为，关注翻译的蛋白质对于准确了解突触和树突如何对环境中的局部变化做出反应至关重要。目前评估细胞或细胞室蛋白质组成变化的现有方法依赖于两个蛋白质组之间的比较。只有当蛋白质的数量足够多，并且比较的蛋白质组之间的蛋白质表达水平有很大差异时，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图谱技术的发展，以分离和鉴定神经元树突中的翻译产物，并确定树突在基础条件和刺激条件下的定量翻译能力。具体地说，我们提出了一种使用人工氨基酸原位标记新合成的树突状蛋白的方法，从而绕过了传统方法的问题。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学问题，无论组织类型或细胞环境。树突状蛋白的变化，与可塑性相关的突触修改，包括成瘾，可以用这项技术来阐明。

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.