Discovery Proteomics Core

发现蛋白质组学核心：综述 发现蛋白质组学核心(DPC)使用了全面的蛋白质图谱技术，这些技术非常 通过互补的生物物理学和脂质组学分析积极发挥杠杆作用。其表达或 翻译后修饰(PTM)在动物或细胞中因暴露于滥用药物而改变 系统将通过包括iTRAQ、SILAC/Silam、Label在内的基于MS的自下而上技术进行识别 自由定量(LFQ)、多维蛋白质识别技术(MudPIT)以及LFQ的耦合 针对SWATH的数据独立采集技术。此外，核心将使用自上而下/中间- Down蛋白质组学方法，以表征完整的蛋白质形式及其PTM。创新样本 准备方法将用于通过激光捕获显微镜更深入地研究神经蛋白质组 (LCM)、基于荧光的细胞学方法(FACS和FANS)和免疫亲和方法 转基因和病毒方法将使细胞类型和细胞器特定的蛋白质组分析成为可能。 蛋白质组表达将通过与目标物合作进行的MRM/SWATH分析来验证 蛋白质组学核心(TPC)。包括等温在内的几种生物物理技术的可用性 微量热(ITC)、静态和动态光散射、圆二色谱(CD)、表面等离子体共振 (SPR)、停流(SF)和不对称流场流动分离(AFFF)将扩展蛋白质图谱 通过定量描述热力学和动力学来分析功能域 基础蛋白质：蛋白质和蛋白质：感兴趣的配体相互作用。在后者中有磷脂酰肌醇 有能力通过与神经功能的相互作用和调节来控制神经功能的几乎每一个方面 神经元蛋白质的活性。结合蛋白质组水平的生物物理和肌醇磷脂分析 分析将提供越来越多的生物系统水平的方法。所有蛋白质图谱数据将被存储 耶鲁大学蛋白质表达数据库(PYED)及其知识库将用于传播这些数据。 此外，与生物信息学和生物统计核心(BBC)一起，DPC将使用RNA测序来 构建实验和细胞类型的特定MS/MS蛋白质参考数据库，该数据库将由 建立PTMome数据库，以提高多肽和蛋白质的识别率。通过采取全面的方法， DPC将为中心调查人员提供识别所需的广泛工具和培训，然后 理解为什么某些蛋白质及其磷脂酰肌醇效应物的差异表达如下 接触精神刺激剂和精神药物。

Discovery Proteomics Core: Summary The Discovery Proteomics Core (DPC) uses a full range of protein profiling technologies that are very positively leveraged by complementary biophysics and lipidomics analyses. Proteins whose expression or posttranslational modifications (PTMs) are altered by exposure to drugs of abuse in animal or cell-based systems will be identified by “bottom-up” MS-based technologies that will include iTRAQ, SILAC/SILAM, Label Free Quantitation (LFQ), Multi-dimensional Protein Identification Technology (MudPIT), and by coupling of LFQ to the data independent acquisition technology of SWATH. In addition, the Core will use Top-down/Middle- down proteomics methodologies to characterize intact proteoforms and their PTMs. Innovative sample preparation approaches will be used to reach deeper into the neural proteome with laser capture microscopy (LCM), fluorescence-based cytometry methods (FACS and FANS), and immuno-affinity methods in conjunction with transgenic and viral approaches that will enable analysis of cell type- and organelle-specific proteomes. Proteome expression will be validated by MRM/SWATH assays carried out in collaboration with the Targeted Proteomics Core (TPC). The availability of several biophysical technologies including isothermal microcalorimetry (ITC), static and dynamic light scattering, circular dichroism (CD), surface plasmon resonance (SPR), stopped-flow (SF), and asymmetric flow field-flow-fractionation (AFFF) will extend protein profiling analyses into the functional domain by quantitatively characterizing the thermodynamics and kinetics that underlie protein:protein and protein:ligand interactions of interest. Among the latter are phosphoinositides that have the ability to control virtually every aspect of neuronal function via their interactions with and modulation of the activities of neuronal proteins. Biophysical and phosphoinositide analyses, combined with proteome level analyses will provide an increasingly biological systems level approach. All protein profiling data will be stored in the Yale Protein Expression Database (YPED) and its Repository will be used to disseminate these data. Further, together with the Bioinformatics and Biostatistics Core (BBC), the DPC will use RNA-sequencing to construct experiment- and cell-type specific MS/MS protein reference databases that will be complemented by a PTMome database to increase peptide and protein identification rates. By taking a holistic approach, the DPC will provide Center investigators with the broad range of tools and training needed to identify, and then to understand why certain proteins and their phosphoinositide effectors are differentially expressed following exposure to psychostimulants and psychotropic drugs.