Proteomic Analysis of Synaptic Vesicle Pools

突触小泡池的蛋白质组学分析

• 批准号: 8571951
• 负责人: ROBERT H EDWARDS
• 金额: $ 23.55万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8571951
• 关键词: Behavior; Biochemical; Cell membrane; Chemicals; Chemistry; Cytolysis; Cytoskeleton; Development; Docking; Electron Microscopy; Exhibits; Foundations; Future; Individual; Label; Magnetism; Mammals; Mass Spectrum Analysis; Membrane Proteins; Molecular; Morphology; Neurons; Organelles; Pattern; Population; Procedures; Process; Property; Proteins; Proteomics; Protocols documentation; Recording of previous events; Recovery; Recycling; Relative (related person); Reporter; Rest; Site; Stable Isotope Labeling; Surface; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptosomes; System; Vesicle; Work; base; nanoparticle; neurotransmitter release; neurotransmitter uptake; optical imaging; public health relevance; research study; response; theories

DESCRIPTION (provided by applicant): Classical studies in the field of synaptic transmission have assumed that neurotransmitter release occurs from a biochemically homogeneous population of synaptic vesicles, but considerable work from many experimental systems has shown that synaptic vesicles belong to pools that differ in their response to stimulation. These observations have given rise to two competing hypotheses, one that the pools are biochemically the same, with differences in behavior strictly stochastic, or extrinsic, reflecting differential association with the cytoskeleton or prior history rather than any intrinsic differences in composition. Alternatively, differences in molecular composition underlie the behavior of different synaptic vesicle pools, and recent work has suggested that the pools may retain their identity after recycling. Although controversial, we have recently shown that different synaptic vesicle proteins respond differently to stimulation, providing some of the first evidence that synaptic vesicle pools differ in composition. However, these experiments involved optical imaging of individual reporter constructs, and understanding how membrane protein composition determines the properties of synaptic vesicles requires a more systematic approach. We will thus label specific synaptic vesicle pools with magnetic nanoparticles strictly on the basis of their response to activity, and determine their composition by quantitative proteomic analysis: Aim 1: Optimize synaptic vesicle recovery from highly purified synaptoneurosomes. Standard procedures fail to recover synaptic vesicles associated with the plasma membrane, so we will optimize synaptic vesicle recovery from synaptoneurosomes using a combination of physical and chemical approaches. Aim 2: Optimize isolation of synaptic vesicles labeled with magnetic nanoparticles during stimulation. We will synthesize small magnetic nanoparticles, and optimize the labeling of different synaptic vesicle pools by different patterns of stimulation. Aim 3: Determine the composition of recycling and resting synaptic vesicle pools by quantitative proteomics using isobaric tag for relative and absolute quantitation (iTRAQ) or stable isotope labeling in mammals (SILAM). Identifying the molecular composition of different synaptic vesicle pools will provide a foundation for future work to explore the functin of the identified components in transmitter release, synapse development and plasticity.

描述（由申请人提供）：突触传播领域的经典研究假设神经递质释放是从生化的突触囊泡的生化均匀群体中发生的，但是许多实验性系统的大量工作表明，突触囊泡属于其对刺激的反应不同的池。这些观察结果引起了两个相互竞争的假设，一个假设在生物化学上是相同的，行为的差异严格是随机的或外部的，反映了与细胞骨架或先前历史的差异关联，而不是组成的任何内在差异。另外，分子组成的差异是不同突触囊泡池的行为的基础，并且最近的工作表明池在回收后可以保留其身份。尽管引起争议，但我们最近表明，不同的突触囊泡蛋白对刺激的反应不同，提供了一些首先证明突触囊泡池在组成方面有所不同。但是，这些实验涉及单个报告基因构建体的光学成像，并了解膜蛋白组成如何确定突触囊泡的特性需要更系统的方法。因此，我们将严格根据其对活性的响应来将特定的突触囊泡池标记为磁性纳米颗粒，并通过定量蛋白质组学分析来确定其组成：目标1：优化从高度纯化的突触型突触中的突触囊泡恢复。标准程序无法恢复与质膜相关的突触囊泡，因此我们将使用物理和化学方法的组合优化突触囊泡从突触中的突触囊泡回收。目标2：优化刺激过程中用磁性纳米颗粒标记的突触囊泡的分离。我们将通过不同的刺激模式合成小磁性纳米颗粒，并优化不同突触囊泡池的标记。 AIM 3：通过定量蛋白质组学，使用同骨标签进行相对和绝对定量（ITRAQ）或稳定的同位素标记（Silam）（Silam），通过定量蛋白质组学来确定回收和静息突触囊泡的组成。识别不同突触囊泡池的分子组成将为将来的工作提供基础，以探索发射机释放，突触发育和可塑性中已鉴定成分的功能。