Targeted Synaptic Proteomics

描述（由申请人提供）：本研究的目标是开发一种定量策略，用于系统监测突触后蛋白的丰度和修饰。突触后致密物（postsynaptic density，PSD）是神经元通讯所必需的特殊膜结构。PSD中蛋白质的动态变化在突触可塑性中起着关键作用。PSD蛋白的失调与多种神经系统疾病如抑郁症、癫痫、物质滥用和神经变性的病因学有关。在PSD中已经鉴定了大约1,000种蛋白质，包括神经递质受体和调节蛋白，但是仍然缺少蛋白质组分的定量、动态视图。关于PSD中的翻译后修饰（例如磷酸化）的调节知之甚少。主要的挑战是如何一致地量化PSD蛋白及其在不同生理和病理条件下的功能修饰。我们假设，有针对性的蛋白质组学将提供一个简单的，大规模的，和敏感的测量蛋白质和修改的PSD。靶向蛋白质组学使用称为选择反应监测（SRM）或多反应监测（MRM）的特定质谱技术。靶向蛋白质组学克服了与传统发现质谱法相关的许多警告，例如有限的再现性，数据冗余和受损的灵敏度。我们将首先获得全面的PSD数据集，然后开发基于SRM的策略来量化核心PSD蛋白以及相关的磷酸化事件。该策略将被彻底测试，以在突触激活的细胞模型中和在亨廷顿病的发展过程中描绘PSD成分。该研究将建立一个基于SRM的策略，有针对性的定量PSD分析。该策略有望大大简化现有的蛋白质组学平台，并将为理解学习，记忆和神经退行性变的分子机制提供重要基础。 公共卫生相关性：突触的分子调节是学习和记忆形成的关键。我们建议开发一种简化的最先进的质谱技术，以定量监测蛋白质的动态和突触后区室的翻译后修饰。这些方法将为研究不同生理和病理条件下突触可塑性提供一种通用方法。

DESCRIPTION (provided by applicant): The goal of this research is to develop a quantitative strategy for systematically monitoring the abundance and modifications of postsynaptic proteins. The postsynaptic density (PSD) is a specialized membrane structure essential for neuronal communication. Dynamic protein changes in the PSD play a pivotal role in synapse plasticity. Dysregulation of PSD proteins has been implicated in the etiology of a variety of neurological disorders, such as depression, epilepsy, substance abuse and neurodegeneration. Approximately 1,000 proteins including neurotransmitter receptors and regulatory proteins have been identified in the PSD, but a quantitative, dynamic view of the protein components is still missing. Much less is known about the modulation of posttranslational modifications (e.g. phosphorylation) in the PSD. The main challenge is how to consistently quantify the PSD proteins and their functional modifications under different physiological and pathological conditions. We hypothesize that targeted proteomics will provide a simple, large-scale, and sensitive measurement of proteins and modifications in the PSD. Targeted proteomics uses a specific mass spectrometry technology termed Selected Reaction Monitoring (SRM) or Multiple Reaction Monitoring (MRM). The targeted proteomics overcomes a number of caveats associated with conventional discovery mass spectrometry, such as limited reproducibility, data redundancy and compromised sensitivity. We will first obtain comprehensive PSD datasets and then develop an SRM-based strategy to quantify core PSD proteins, as well as associated phosphorylation events. The strategy will be thoroughly tested to profile PSD components in a cellular model of synaptic activation and during the development of Huntington disease. The study will establish an SRM-based strategy for targeted quantitative PSD analysis. The strategy is expected to greatly simplify current proteomics platform, and will provide crucial basis for understanding molecular mechanisms of learning, memory and neurodegeneration. PUBLIC HEALTH RELEVANCE: Molecular regulation of synapse is the key to the formation of learning and memory. We propose to develop a simplified state-of-the-art mass spectrometry technology to quantitatively monitor the dynamics of proteins and posttranslational modifications in the postsynaptic compartment. The methods will provide a generic approach for investigating the synaptic plasticity under diverse physiological and pathological conditions.