Accelerated Protein Signaling Signatures

加速蛋白质信号传导特征

• 批准号: 8725256
• 负责人: Jacob David Jaffe
• 金额: $ 50.3万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-16 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8725256
• 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计划旨在从细胞扰动中得出分子特征。细胞 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，是与转录分析的补充。该建议详细介绍了高度的发展 信息内容多重质谱法对查询丝氨酸和苏氨酸信号传导的测定 途径。还原主义的方法，在该方法下，多种磷酸化位点的自然相关性 阐明了不同的细胞条件，将用于在表示细胞方面产生效率 状态。为此，在变化下的数量适度 将使用质谱（MS）获得条件。从这些数据中，数量有限 将提取代表性的磷酸肽 - 减少的表示集 - 其水平和水平 石化的图表将用于响应扰动而捕获特征。随后，必要的 将采购试剂以设计目标质谱分析（多重反应 监测，或MRM-MS），以量化在多路复用（〜100 plex）中的减少表示中的肽 时尚。可变性，可重复性，检测和量化限制以及最终测定成本将为 测量。该测定法的多实验室实施也将得到证明。与这些平行 努力，已建立的开放访问蛋白质组学软件天际线将扩展以提供标准 住房的手段重要测定参数，从减少表示集中获取的数据 MRM-MS测定，并集成QA/QC指标以确定测定性能。天际线也将是 适应MRM-MS方法和数据的实验室间交换，我们将开发公众 数据库将整合来自多个Lincs实验室的测定结果。最终产品将很高 信息内容多重MS分析称为“加速蛋白质信号签名”和 信息基础架构支持包含所有必要信息的社区资源和 以协作方式在多个蛋白质组学实验室进行实际实施的工具。