Comprehensive Synaptome Proteomics Targeting Protein Expression and PTMs in HD

针对 HD 中蛋白质表达和 PTM 的综合突触组蛋白质组学

基本信息

批准号：
9149037
负责人：
Lisa M Ellerby
金额：
$ 27.57万
依托单位：
BUCK INSTITUTE FOR RESEARCH ON AGING
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-30 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9149037
关键词：
Acetylation Address Affect Affinity Aftercare Alzheimer&apos s Disease Animals Basal Ganglia Bioinformatics Biological Models Biology Brain Brain region Cell model Cells Child Chorea Complex Corpus striatum structure Cytoplasm Data Data Analyses Data Set Development Disease Disease Progression Dopamine Dopamine D1 Receptor Dopamine D2 Receptor Endocytosis Exocytosis Functional disorder Genes Genetic Crosses Goals Heterogeneity Histone Deacetylase Human Huntington Disease Huntington gene Impaired cognition In Vitro Individual Inherited Ions Knock-in Mouse Knowledge Label Lead Libraries Lysine Mass Spectrum Analysis Measurement Measures Mediating Membrane Mental disorders Methodology Methods Mitochondria Modeling Movement Disorders Mus Mutation Neurodegenerative Disorders Neuronal Dysfunction Neurons Norepinephrine Parents Parkinson Disease Pathology Pathway interactions Peptides Pharmaceutical Preparations Phenotype Phosphorylation Physiological Play Post-Translational Protein Processing Preparation Presynaptic Terminals Proteins Proteome Proteomics Q-Type Calcium Channels Recruitment Activity Regulation Reporter Reporting Role SNAP receptor Sampling Scanning Serotonin Site Small Interfering RNA Synapses Synaptic Vesicles Synaptosomes Technology Testing Time Tissue Sample Tissues Trinucleotide Repeats VAMP-2 Vesicle base cell type density disease phenotype experience fly functional status gamma-Aminobutyric Acid improved induced pluripotent stem cell inhibitor/antagonist knock-down middle age motor symptom mouse model mutant nerve stem cell neurotransmission neurotransmitter release novel novel strategies novel therapeutics overexpression phosphatase inhibitor polyglutamine promoter protein complex protein expression public health relevance research study synaptic function synaptosomal-associated protein 25 syntaxin 1A

项目摘要

DESCRIPTION (provided by applicant): An essential goal of quantitative proteomics is to understand how proteins in cells and tissues change in their expression levels and posttranslational modification (PTM) status, ideally with knowledge of their spatial and temporal reorganization, protein interaction networks and functional status. These attributes are currently impossible to obtain in any single experiment, and thus require the development and implementation of multiple technologies. A critical complicating factor in mass spectrometry-based proteomic methods is how to sample tissues, especially ones that are inherently heterogeneous. The brain is particularly challenging as there are dozens of defined subregions, and where each region contains combinations of neuronal, glial and other cell types. This underlying heterogeneity can greatly complicate and obscure data interpretation in tissue proteomics, especially when one is tasked with identifying neuronal dysfunction in diseases such as Parkinson's, Alzheimer's and Huntington's disease. Methods for preparing a well-defined fraction from brain synapses, the synaptosome, has been available for some time, although improvements have been made in recent years with regards to increased efficiency and purity. Synaptosomes contain components of both the pre-and post-synaptic terminal, including mitochondria, vesicles, cytoplasm and post-synaptic density, and are therefore still a relatively complex cellular/synaptic fraction. In this application, we propose to tackle the inherent complexity of synaptosomes using mass spectrometry-based proteomic methods that will improve sampling efficiency and quantitative methodology, as well as provide information on temporal dynamics and PTMs. Proteomic sampling efficiency of the synaptome will be increased by generating a comprehensive spectral library and then using a new SWATH acquisition approach to obtain fragment and primary ion data on all ionizable analytes. For improved quantitation, novel label-free methods will integrate both primary scan (MS1 Filtering) and collisional induced fragment (MS2) ion intensity data. Affinity enrichment methods will target phosphorylation and lysine-acetylation, both important in synapse function and regulation. As we have extensive experience in mouse models of Huntington's disease (HD), we propose to use an Htt-polyQ expanded knockin mouse, zQ175 compared to littermate controls (B6/J background), as our model system. Synaptosomes isolated from the cortex and striatum will be examined using these proteomic strategies, as these brain regions are known to be effected in HD. The striatal and cortical proteome will lay the groundwork for the defining the subtypes of synaptosomes that will be purified from reporter zQ175 mice (genetic crosses). We will also use this data set to identify modifiers of HD disease progression using induced pluripotent stem cell models of HD and siRNA knockdown strategies.

描述（由申请人提供）：定量蛋白质组学的一个基本目标是了解细胞和组织中蛋白质的表达水平和翻译后修饰（PTM）状态如何变化，最好了解其空间和时间重组、蛋白质相互作用网络和功能这些属性目前不可能在任何单一实验中获得，因此需要开发和实施多种技术，基于质谱的蛋白质组学方法的一个关键复杂因素是如何对组织进行采样，特别是那些组织。大脑具有固有的异质性，因为它有数十个明确的子区域，并且每个区域都包含神经细胞、神经胶质细胞和其他细胞类型的组合，这种潜在的异质性会使组织蛋白质组学中的数据解释变得非常复杂和模糊，尤其是当一个人承担任务时。帕金森病、阿尔茨海默病和亨廷顿病等疾病中的神经元功能障碍，从大脑突触中制备明确的部分（突触体）的方法已经存在一段时间了。尽管近年来在提高效率和纯度方面取得了进步，但突触体含有突触前和突触后末端的成分，包括线粒体、囊泡、细胞质和突触后密度，因此仍然是相对复杂的细胞。在本应用中，我们建议使用基于质谱的蛋白质组学方法来解决突触体固有的复杂性，这将提高采样效率和定量方法，并提供有关时间动态和信息的信息。通过生成综合谱库，然后使用新的 SWATH 采集方法获取所有可电离分析物的碎片和初级离子数据，将提高突触组的蛋白质组采样效率。为了改进定量，新型无标记方法将整合两种初级离子。扫描（MS1 过滤）和碰撞诱导碎片（MS2）离子强度数据将针对突触中重要的磷酸化和赖氨酸乙酰化。由于我们在亨廷顿病 (HD) 小鼠模型方面拥有丰富的经验，因此我们建议使用 Htt-polyQ 扩增的敲入小鼠 zQ175 与同窝广泛对照（B6/J 背景）作为我们分离的模型系统。将使用这些蛋白质组学策略来检查皮质和纹状体的蛋白质组，因为已知这些大脑区域在 HD 中受到影响，纹状体和皮质蛋白质组将为定义奠定基础。将从报告 zQ175 小鼠中纯化的突触体亚型（遗传杂交）我们还将利用 HD 诱导多能干细胞模型和 siRNA 敲低策略，使用该数据集来识别 HD 疾病进展的修饰因子。