Comprehensive Synaptome Proteomics Targeting Protein Expression and PTMs in HD

 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，理想情况下了解其空间和时间重组、蛋白质相互作用网络和功能状态。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。在基于质谱的蛋白质组学方法中，一个关键的复杂因素是如何对组织进行采样，特别是那些固有异质性的组织。大脑特别具有挑战性，因为有几十个定义的子区域，每个区域都包含神经元，神经胶质和其他细胞类型的组合。这种潜在的异质性可以极大地复杂化和模糊组织蛋白质组学中的数据解释，特别是当一个人的任务是确定神经元功能障碍的疾病，如帕金森氏症，阿尔茨海默氏症和亨廷顿氏病。用于从脑突触制备明确定义的级分（突触体）的方法已经可用了一段时间，尽管近年来在提高效率和纯度方面进行了改进。突触体含有突触前和突触后末端的组分，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/突触部分。 在此应用中，我们建议使用基于质谱的蛋白质组学方法来解决突触体的固有复杂性，这将提高采样效率和定量方法，并提供有关时间动态和PTM的信息。通过生成全面的光谱库，然后使用新的SWATH采集方法获得所有可电离分析物的片段和初级离子数据，将提高突触体的蛋白质组学采样效率。为了改进定量，新的无标记方法将整合初级扫描（MS 1过滤）和碰撞诱导碎片（MS 2）离子强度数据。亲和富集方法将靶向磷酸化和赖氨酸乙酰化，这两者在突触功能和调节中都很重要。由于我们在亨廷顿氏病（HD）的小鼠模型中具有丰富的经验，我们建议使用与同窝对照（B6/J背景）相比的Htt-polyQ扩增敲入小鼠zQ 175作为我们的模型系统。将使用这些蛋白质组学策略检查从皮质和纹状体分离的突触体，因为已知这些脑区域在HD中受到影响。纹状体和皮质蛋白质组将为定义将从报告基因zQ 175小鼠（遗传杂交）中纯化的突触体亚型奠定基础。我们还将使用该数据集来鉴定使用HD和siRNA敲低策略的诱导多能干细胞模型的HD疾病进展的修饰剂。

 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.