Accelerated Protein Signaling Signatures

描述（由申请人提供）：LINCS计划旨在获得由细胞扰动产生的分子特征。通过调节蛋白质磷酸化的细胞信号传导是细胞对刺激反应的重要组成部分，并且与转录谱互补。该提案详细介绍了一种高信息含量的基于多重质谱的检测方法，以查询丝氨酸和苏氨酸信号通路的发展。一个还原论的方法，从而阐明不同的细胞条件下的多个磷酸化位点的自然相关性，将被用来创建效率代表细胞状态。为了实现这一点，将使用质谱法（MS）获得不同条件下的适度数量的定量全局磷酸化蛋白质组学图谱。从这些数据中，将提取有限数量的代表性磷酸肽-简化的代表集-其水平和化学计量将用于捕获响应扰动的签名。随后，将采购必要的试剂，以允许设计靶向质谱测定（多反应监测或MRM-MS），从而以多重（~100重）方式定量简化代表集中的肽。将测量变异性、再现性、检测限和定量限以及最终测定成本。还将展示该检测试剂盒的多实验室实施。在这些努力的同时，将扩展已建立的开放获取蛋白质组学软件Skyline，以提供容纳重要测定参数的标准方法，处理从简化代表集MRM-MS测定获得的数据，并整合QA/QC指标以确定测定性能。Skyline还将进行调整，以允许实验室间交换MRM-MS方法和数据，我们将开发一个公共数据库，该数据库将整合来自多个LINCS实验室的检测结果。最终产品将是一个高信息含量的多重MS分析，称为“加速蛋白质信号转导签名”和信息学基础设施，以支持社区资源，其中包含所有必要的信息和工具，用于以协作方式在多个蛋白质组学实验室中实际实施。 公共卫生相关性：这些实验的发展将使我们能够确定人类细胞对药物治疗的反应，而时间和成本是可比方法的一小部分。这些实验提供的信息将为我们提供潜在新疗法的早期指标，并帮助我们区分具有预期效果的药物和可能具有不良副作用的药物。

DESCRIPTION (provided by applicant): The LINCS program seeks to derive molecular signatures resultant from cellular perturbation. Cellular signaling through modulation of protein phosphorylation is an important component of the 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 of the 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 of the necessary information and tools for its practical implementation across multiple proteomics laboratories in a collaborative manner. PUBLIC HEALTH RELEVANCE: Development of these experiments will allow us to determine how human cells respond to drug treatment for a fraction of the time and cost of comparable methods. The information provided from these experiments will give us early indicators of potential new therapies and help us differentiate drugs that have desired effects from those that might have undesirable side effects.