Discovery Proteomics Core

发现蛋白质组学核心

• 批准号: 10025467
• 负责人: TuKiet T Lam
• 金额: $ 76.73万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10025467
• 关键词: Binding; Bioinformatics; Biological; Biological Assay; Biometry; Biophysics; Biotechnology; Brain; Brain region; Carrier Proteins; Cells; Chemicals; Circular Dichroism; Collaborations; Complex; Computer software; Computers and Advanced Instrumentation; Custom; Data; Data Analyses; Data Set; Databases; Development; Digestion; Drug Addiction; Ensure; Enzymes; Epigenetic Process; Experimental Designs; Exposure to; Field Flow Fractionation; Fluorescence Resonance Energy Transfer; Fractionation; Generations; Glycopeptides; Immunologics; Individual; Information Dissemination; Ions; Kinetics; Label; Lectin; Libraries; Ligands; Lipids; Liquid substance; Mass Spectrum Analysis; Medical Libraries; Membrane; Metabolism; Methodology; Methods; Monitor; National Institute of Drug Abuse; Nucleic Acids; Organelles; Output; Peptides; Phosphatidylinositols; Phosphopeptides; Post-Translational Modification Site; Post-Translational Protein Processing; Preparation; Proteins; Proteome; Proteomics; Qi; Reaction; Recombinant Proteins; Research; Research Personnel; Resolution; Sampling; Scaffolding Protein; Scheme; Signal Transduction; Site; Software Tools; Spottings; Students; Surface Plasmon Resonance; System; Techniques; Technology; Thermodynamics; Time; Tissues; Training; Vendor; Visualization; Work; addiction; base; biophysical analysis; cell type; combinatorial; design; differential expression; drug action; drug of abuse; glycosylation; histone modification; holistic approach; improved; innovation; interest; light scattering; lipid transport; microcalorimetry; neuroproteomics; neurotransmission; overexpression; polypeptide; programs; protein complex; protein expression; protein function; protein profiling; response; small molecule; titanium dioxide; tool; transcriptome sequencing

The Discovery Proteomics Core (DPC) uses cutting edge sample preparation, state-of-the-art proteomic and complementary recombinant protein expression, biophysical, and lipidomics technologies to analyze adaptive changes in neuronal signal transduction mechanisms that occur in response to drugs of abuse. Aim 1 uses discovery proteomics and Data-Independent Acquisition (DIA) from the Targeted Proteomics Core (TPC) to identify proteins whose expression or posttranslational modifications (PTMs) are altered in cell-based systems, tissue from discrete brain regions, single cell types and their organelles isolated with LCM or FACS, or protein complexes isolated by proximity labeling or other approaches. To improve peptide identification from searching databases with MS/MS spectra, we will use DIA and ETD-DIA and we will collaborate with the Biostatistics and Bioinformatics Core (BBC) to enable MS/MS searches of RNA-sequencing-predicted proteomes. We will build peptide MS/MS spectral libraries to support DIA assays. Aim 2 uses immunological and chemical approaches to enrich for peptides and proteins containing PTMs to facilitate their identification. In collaboration with TPC, the DPC will integrate PTM analyses into DIA, including use of ETD for glycosylation and “Middle-down” analyses, and use Parallel Reaction Monitoring by TPC to validate differentially expressed proteins and PTMs. Aim 3 uses “Middle-down” analyses to identify the multiple PTMs that occur in individual proteoforms such as the combinatorial epigenetic changes in histone modification. In Aim 4 we will collaborate with the BBC to streamline data output to improve visualization of quantitative protein PTM changes, and will improve existing and design new tools to carry out more advanced data analyses. In Aim 5 we will overexpress and purify recombinant proteins for our investigators and use isothermal microcalorimetry, static/dynamic light scattering, circular dichroism, surface plasmon resonance, stopped-flow, and asymmetric flow field-flow-fractionation to extend protein profiling into the functional domain by quantitatively determining the thermodynamics and kinetics that underlie protein:protein and protein:ligand interactions. In Aim 6 we will quantify phosphoinositide lipids in brain circuits involved in addiction and provide innovative cell-free assays for studying lipid transport proteins that function at membrane contact sites. In collaboration with TPC, we will build a DIA assay for the phosphoinositide interactome. In Aim 7, we will provide training in experimental design, sample preparation, and use of softwares for analysis and interpretation of data from MS, biophysical, and phosphoinositide analyses. The DPC will collaborate with Center investigators and will ensure that the Center's research is supported by the most advanced instrumentation and biotechnologies. 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 PTMs and phosphoinositide effectors are differentially expressed following exposure to drugs of abuse.

Discovery 蛋白质组学核心 (DPC) 采用尖端的样品制备、最先进的蛋白质组学和 互补的重组蛋白表达、生物物理和脂质组学技术来分析适应性 因滥用药物而发生的神经信号转导机制的变化。目标 1 使用 从靶向蛋白质组学核心 (TPC) 到发现蛋白质组学和数据独立采集 (DIA) 识别其表达或翻译后修饰（PTM）在基于细胞的系统中发生改变的蛋白质， 使用 LCM 或 FACS 或蛋白质分离的离散脑区域组织、单细胞类型及其细胞器 通过邻近标记或其他方法分离的复合物。通过搜索改进肽鉴定 具有 MS/MS 谱图的数据库，我们将使用 DIA 和 ETD-DIA，我们将与生物统计和 生物信息学核心 (BBC)，可对 RNA 测序预测的蛋白质组进行 MS/MS 搜索。我们将建设 支持 DIA 测定的肽 MS/MS 谱库。目标 2 使用免疫学和化学方法 富集含有 PTM 的肽和蛋白质，以促进其识别。与 TPC 合作， DPC 将把 PTM 分析整合到 DIA 中，包括使用 ETD 进行糖基化和“Middle-down” 分析，并使用 TPC 的平行反应监测来验证差异表达的蛋白质和 PTM。 目标 3 使用“中下”分析来识别单个蛋白质形式中出现的多个 PTM，例如 组蛋白修饰的组合表观遗传变化。在目标 4 中，我们将与 BBC 合作 简化数据输出，以改善定量蛋白质 PTM 变化的可视化，并将改善现有的 并设计新工具来进行更高级的数据分析。在目标 5 中，我们将过度表达和纯化 为我们的研究人员提供重组蛋白，并使用等温微量热法、静态/动态光散射、 圆二色性、表面等离子体共振、停流和不对称流场流分级 通过定量确定热力学和功能，将蛋白质分析扩展到功能域 蛋白质：蛋白质和蛋白质：配体相互作用的动力学。在目标 6 中，我们将量化磷酸肌醇 与成瘾有关的大脑回路中的脂质，并为研究脂质转运提供创新的无细胞测定 在膜接触位点发挥作用的蛋白质。我们将与 TPC 合作，构建 DIA 检测方法 磷酸肌醇相互作用组。在目标 7 中，我们将提供实验设计、样品制备、 以及使用软件分析和解释来自 MS、生物物理和磷酸肌醇的数据 分析。 DPC 将与中心研究人员合作，并确保该中心的研究是 由最先进的仪器和生物技术支持。通过采取整体方法，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 了解为什么某些蛋白质及其 PTM 和磷酸肌醇效应物的表达存在差异 接触滥用药物后。