Targeted Proteomics Core

靶向蛋白质组学核心

• 批准号: 8935162
• 负责人: CHRISTOPHER MICHAEL COLANGELO
• 金额: $ 34.27万
• 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: 2004-08-23 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8935162
• 关键词: Address; Alleles; Alternative Splicing; Archives; Award; Binding; Bioinformatics; Biological; Biological Assay; Biological Markers; Biostatistics Core; Brain; Brain region; Cell Extracts; Cell Nucleus; Cells; Collaborations; Complement; Computer Simulation; Data; Data Set; Databases; Development; Digestion; Event; Experimental Designs; Exposure to; Genetic Transcription; Genomics; Goals; Housing; Human; Hybrids; Interest Group; Ions; Label; Liquid Chromatography; Liquid substance; Literature; Mass Spectrum Analysis; Methods; Mitochondria; Modeling; Modification; Mus; National Institute of Drug Abuse; Neurons; Organelles; Organism; Peptide Fragments; Peptide Library; Peptides; Phosphatidylinositols; Phosphopeptides; Phosphoproteins; Phosphorylation Site; Post-Translational Regulation; Preparation; Property; Proteins; Proteome; Proteomics; Protocols documentation; RNA Editing; Rattus; Relative (related person); Research; Research Personnel; Resolution; Running; Sampling; Shotguns; Signal Transduction; Synapses; Synaptic Vesicles; Techniques; Technology; Time; Tissue Extracts; Tissues; Training; cell type; comparative; density; design; drug of abuse; innovation; instrument; interest; mRNA Expression; mass spectrometer; member; multiple reaction monitoring; neurotransmission; next generation; novel strategies; protein expression; protein profiling; response; selective expression; transcriptome sequencing

Targeted Proteomics Core: Summary The Targeted Proteomics Core designs and implements targeted assays that have been used increasingly by Center investigators to rapidly quantitate and validate potential protein biomarkers related to their studies of neuronal signaling and the actions of drugs of abuse. By utilizing our in-house targeted proteome workflows for both MRM and data-independent (DIA) analysis, we propose to collaborate with NIDA Center investigators, and the Discovery Proteomics and Bioinformatics and Biostatistics Cores, to accurately quantitate either protein expression or their modifications in selected sub-groups of interest. Fully consistent with the overall goals of the Center, targeted proteomic approaches will be used in an innovative manner to systematically address the inherent challenges of single cell type analysis through the ability to design MRM and DIA assays for subcellular organelles and sub-proteomes. Targeted proteomic approaches will also be used to leverage RNA-seq data from selected brain regions or neuronal cell types, for example, in directed analysis of specific proteins or groups of related proteins that are selectively expressed or regulated in a specific cell type. Such targeted approaches will be unique to the TPC and will have a major impact on the ability of Center investigators to carry out innovative research using state-of-the-art approaches to study the action of drugs of abuse. Specifically, we will leverage our optimized MRM workflow by developing highly sensitive MRM assays to examine the proteomes of specific organelles (e.g., nuclei, mitochondria, synaptic vesicles), sub-cellular fractions or partially enriched samples from single types of neuronal cells. We will also expand our current rat/mouse brain targeted MRM PSD proteome assay from 112 proteins to 200 proteins. In addition, we will integrate and expand high resolution DIA acquisition into our next generation large scale targeted proteome assays directed at sub-proteomes from specific types of neurons. Other projects include implementation of a DIA assay to quantify as many as possible of the ~400 proteins that have been identified as being members of the human phosphoinositide-binding proteome. DIA analysis has the potential to change the proteomic landscape since each sample only needs to be run once, and then all peptide fragments can retrospectively be identified and quantified. To advance these studies, we will build a library of peptide biomarkers that will be posted publically on our YPED database and that can be used by multiple investigators in the Center and elsewhere who analyze basic signal transduction mechanisms in the brain, or who study the effects of exposure to drugs of abuse on these signaling mechanisms. Importantly, we will continue to train Neuroproteomics Center members in mass spectrometric techniques including experimental design, sample preparation (e.g. tissues, cells) and handling, digestion protocols, interpretation of MRM and DIA spectra so they can optimally utilize the advanced technologies available in the Center.

靶向蛋白质组学核心：总结 靶向蛋白质组学核心设计并实施了越来越多地使用的靶向测定 由中心研究人员快速定量和验证与其研究相关的潜在蛋白质生物标志物 神经信号传导和滥用药物的作用。通过利用我们内部的目标蛋白质组工作流程 MRM 和数据独立 (DIA) 分析，我们建议与 NIDA 中心研究人员合作， 以及 Discovery 蛋白质组学、生物信息学和生物统计学核心，以准确定量 蛋白质表达或其在选定的感兴趣亚组中的修饰。与整体完全一致 为了实现该中心的目标，将以创新的方式使用有针对性的蛋白质组学方法来系统地 通过设计 MRM 和 DIA 检测的能力解决单细胞类型分析的固有挑战 用于亚细胞器和亚蛋白质组。靶向蛋白质组学方法也将用于利用 来自选定大脑区域或神经元细胞类型的 RNA-seq 数据，例如，在特定的定向分析中 在特定细胞类型中选择性表达或调节的蛋白质或相关蛋白质组。这样的 有针对性的方法将是 TPC 所独有的，并将对中心的能力产生重大影响 研究人员使用最先进的方法进行创新研究，以研究药物的作用 虐待。具体来说，我们将通过开发高灵敏度的 MRM 检测来利用我们优化的 MRM 工作流程 检查特定细胞器（例如细胞核、线粒体、突触小泡）、亚细胞的蛋白质组 来自单一类型神经元细胞的级分或部分富集样本。我们还将扩大现有的 大鼠/小鼠脑靶向 MRM PSD 蛋白质组测定从 112 种蛋白质到 200 种蛋白质。此外，我们将 将高分辨率 DIA 采集整合并扩展至我们的下一代大规模靶向蛋白质组中 针对特定类型神经元的亚蛋白质组的测定。其他项目包括实施 DIA 测定尽可能多地量化已确定为以下成员的约 400 种蛋白质 人类磷酸肌醇结合蛋白质组。 DIA 分析有可能改变蛋白质组学 景观，因为每个样本只需要运行一次，然后所有肽片段都可以追溯 识别并量化。为了推进这些研究，我们将建立一个肽生物标志物库， 公开发布在我们的 YPED 数据库上，可供中心的多名调查人员使用 其他人分析大脑中的基本信号转导机制，或研究以下因素的影响： 接触滥用药物对这些信号机制的影响。重要的是我们会继续训练 神经蛋白质组学中心成员从事质谱技术，包括实验设计、样品 准备（例如组织、细胞）和处理、消化方案、MRM 和 DIA 光谱的解释，以便 他们可以最佳地利用该中心现有的先进技术。