Analysis of the Dynamic Synaptic Proteome

描述（由申请人提供）：突触是动态的，就其突触传递的幅度、其形态结构和其蛋白质组分而言。由蛋白质合成、降解、分布和翻译后修饰的变化介导的突触蛋白质组的动态变化影响突触功能和突触可塑性的能力。尽管认识到突触的生物学重要性， 蛋白质动力学、富集和鉴定动态突触蛋白质组的方法还不是神经科学研究的组成部分。主要在原核生物和非神经元细胞中进行的研究已经证明了使用将非规范氨基酸掺入新合成的蛋白质中，然后进行生物正交点击化学以用探针（如生物素）标记非规范氨基酸标记的蛋白质的前景，所述探针可用于纯化和鉴定新合成的蛋白质。我们提出的实验，以优化使用非典型的氨基酸掺入和生物正交点击化学在神经元细胞培养，并在视觉系统的完整的大鼠和非洲爪蟾蝌蚪，以证明代谢标记的可行性，以确定和量化的动态组件的突触蛋白质组。这些实验是为了证明比较不同条件和不同实验系统中突触蛋白合成的原理。

DESCRIPTION (provided by applicant): Synapses are dynamic, with respect to their magnitude of synaptic transmission, their morphologic structure and their protein components. Dynamic changes in the synaptic proteome, mediated by changes in protein synthesis, degradation, distribution and post- translational modifications affect synaptic function and the capacity for synaptic plasticity. Despite the appreciation of the biological importance of synaptic protein dynamics, methods to enrich for and identify the dynamic synaptic proteome are not yet an integral part of neuroscience research. Studies conducted largely in prokaryotes and non-neuronal cells have demonstrated the promise of using the incorporation of non- canonical amino acids into newly synthesized proteins followed by bio-orthogonal click chemistry to tag the non-canonical amino acid labeled protein with probes, such as biotin, that can be used for purification and identification of newly synthesized proteins. We propose experiments to optimize the use of non-canonical amino acid incorporation and bio-orthogonal click chemistry in neuronal cell cultures, and in the visual system of intact rats and Xenopus tadpoles to demonstrate the feasibility of metabolic labeling to identify and quantify dynamic components of the synaptic proteome. These experiments are meant to represent proof of principal for comparing synthesis of synaptic proteins between different conditions and in different experimental systems.