Discovery Proteomics Core

发现蛋白质组学核心

• 批准号: 10204999
• 负责人: TuKiet T Lam
• 金额: $ 65.13万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10204999
• 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; lipidomics; 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 Proteomics Core（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、生物物理学和脂质组学技术来分析适应性 神经元信号转导机制的变化，发生在响应滥用药物。Aim 1使用 发现蛋白质组学和数据独立采集（DIA）从靶向蛋白质组学核心（TPC）， 鉴定其表达或翻译后修饰（PTM）在基于细胞的系统中改变的蛋白质， 来自离散脑区域的组织，用LCM或FACS分离的单细胞类型及其细胞器，或蛋白质 通过邻近标记或其他方法分离复合物。从搜索中改进肽鉴定 数据库与MS/MS光谱，我们将使用DIA和ETD-DIA，我们将与生物统计学和 生物信息学核心（BBC），使RNA测序预测的蛋白质组的MS/MS搜索。我们将建立 肽MS/MS光谱库以支持DIA测定。AIM 2使用免疫学和化学方法 富集含有PTM的肽和蛋白质，以促进其鉴定。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“中-下” 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。 目的3使用“中间-向下”分析来鉴定在单个蛋白质型中发生的多个PTM，例如 组蛋白修饰中的表观遗传学组合变化。在Aim 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 并设计新的工具来进行更先进的数据分析。在目标5中，我们将过度表达和纯化 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， 通过定量测定热力学将蛋白质谱扩展到功能域， 蛋白质：蛋白质和蛋白质：配体相互作用的动力学基础。在目标6中，我们将定量磷酸肌醇 脂质在大脑回路中参与成瘾，并提供创新的无细胞测定研究脂质转运 在膜接触位点发挥作用的蛋白质。与TPC合作，我们将建立一个DIA测定， 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MS、生物物理和磷酸肌醇数据 分析。DPC将与中心的研究人员合作，并将确保中心的研究 由最先进的仪器和生物技术支持。通过采取整体方法，DPC 将为中心研究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PTM和磷酸肌醇效应子的差异表达 在接触药物滥用后。