Accelerated Protein Signaling Signatures

加速蛋白质信号传导特征

• 批准号: 8643325
• 负责人: Jacob David Jaffe
• 金额: $ 21.47万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8643325
• 关键词: Adoption; Adverse effects; Antibodies; Biological Assay; Bypass; Cell Count; Cell Line; Cells; Communities; Computer software; Data; Databases; Detection; Development; Event; Goals; Hour; Housing; Human; Informatics; Institutes; Instruction; Laboratories; Libraries; Maps; Mass Spectrum Analysis; Measurement; Measures; Messenger RNA; Methods; Metric; Molecular Profiling; Pathway interactions; Peptides; Performance; Pharmaceutical Preparations; Phosphopeptides; Phosphorylation; Phosphorylation Site; Proteins; Proteomics; Quality Control; Reagent; Reproducibility; Research Infrastructure; Resources; Serine; Serine/Threonine Phosphorylation; Signal Pathway; Signal Transduction; Signaling Protein; Site; Stimulus; Threonine; Threonine Phosphorylation Site; Time; Variant; assay development; base; computerized data processing; cost; design; instrument; instrumentation; mass spectrometer; member; multiple reaction monitoring; programs; research study; response; stoichiometry; synthetic peptide; tool

The LINCS program seeks to derive molecular signatures resultant from cellular perturbation. Cellular signaling through modulation of protein phosphorylation is an important component ofthe cellular response to stimuli, and is complementary to transcriptional profiling. This proposal details the development of a high information content multiplex mass spectrometry-based assay to query serine and threonine signaling pathways. A reductionist approach, whereby the natural correlations of multiple phosphorylation sites under disparate cellular conditions are elucidated, will be used to create efficiencies in representing the cellular state. To achieve this, a modest number of quantitative global phosphoproteomic profiles under varying conditions will be obtained using mass spectrometry (MS). From these data, a limited number of representative phosphopeptides will be extracted - the reduced representation set - whose levels and stoichiometries will serve to capture signatures in response to perturbation. Subsequently, the necessary reagents will be procured to allow the design of targeted mass spectrometry assays (multiple reaction monitoring, or MRM-MS) to quantify the peptides in the reduced representation set in a multiplex (~100-plex) fashion. Variability, reproducibility, limits of detection and quantification, and final assay cost will be measured. Multi-laboratory implementation ofthe assay will also be demonstrated. In parallel to these efforts, the established open access proteomics software Skyline will be extended to provide a standard means of housing important assay parameters, processing data acquired from reduced representation set MRM-MS assays, and integrating QA/QC metrics to determine assay performance. Skyline will also be adapted to allow for inter-laboratory exchange of MRM-MS methods and data, and we will develop a public database that will integrate assay results from multiple LINCS laboratories. The final product will be a high information content multiplex MS assay called the "Accelerated Protein Signaling Signature" and the informatics infrastructure to support a community resource that contains all ofthe necessary information and tools for its practical implementation across multiple proteomics laboratories in a collaborative manner.

LINCS 项目旨在获取细胞扰动产生的分子特征。蜂窝网络 通过调节蛋白质磷酸化的信号传导是细胞反应的重要组成部分 刺激，并且与转录分析互补。该提案详细介绍了高 信息内容基于多重质谱分析来查询丝氨酸和苏氨酸信号传导 途径。还原论方法，其中多个磷酸化位点的自然相关性 阐明了不同的细胞条件，将用于提高代表细胞的效率 状态。为了实现这一目标，需要在不同的条件下进行适量的定量全球磷酸化蛋白质组谱分析。 条件将使用质谱（MS）获得。从这些数据来看，数量有限 将提取代表性磷酸肽 - 简化的表示集 - 其水平和 化学计量将用于捕获响应扰动的特征。随后，必要的 将采购试剂以允许设计靶向质谱分析（多重反应 监测或 MRM-MS）以量化多重（约 100 重）中简化表示集中的肽 时尚。变异性、再现性、检测和定量限制以及最终检测成本将 测量。还将演示该测定的多实验室实施。与这些并行 经过努力，已建立的开放获取蛋白质组学软件 Skyline 将得到扩展，以提供标准 容纳重要测定参数、处理从简化表示集获取的数据的方法 MRM-MS 检测，并集成 QA/QC 指标以确定检测性能。天际线也将 适合允许实验室间交换 MRM-MS 方法和数据，我们将开发一个公共 数据库将整合多个 LINCS 实验室的检测结果。最终产品将是一个高 信息内容多重 MS 测定称为“加速蛋白质信号传导特征”和 支持社区资源的信息学基础设施，其中包含所有必要的信息和 以协作方式在多个蛋白质组学实验室实际实施的工具。