Accelerated Protein Signaling Signatures

描述（由申请人提供）：LINCS项目寻求从细胞扰动中获得分子特征。通过蛋白质磷酸化调节的细胞信号是细胞对刺激反应的重要组成部分，并且是转录谱的补充。该提案详细介绍了一种基于多重质谱的高信息含量分析方法的发展，以查询丝氨酸和苏氨酸信号通路。一种简化的方法，即在不同的细胞条件下阐明多个磷酸化位点的自然相关性，将用于在表示细胞状态时创造效率。为了实现这一目标，将使用质谱法（MS）在不同条件下获得少量定量的全球磷蛋白质组学谱。从这些数据中，将提取有限数量的代表性磷酸肽-减少的表示集-其水平和化学计量将用于捕获响应扰动的特征。随后，将采购必要的试剂，以便设计靶向质谱分析（多重反应监测，或MRM-MS），以多重（~100-plex）方式定量减少的表示集中的肽。将测量变异性、可重复性、检测和定量限以及最终分析成本。还将演示该分析的多实验室实施。与此同时，已建立的开放获取蛋白质组学软件Skyline将被扩展，以提供一种容纳重要分析参数的标准手段，处理从减少表示集MRM-MS分析中获得的数据，并整合QA/QC指标来确定分析性能。Skyline还将允许实验室间交换MRM-MS方法和数据，我们将开发一个公共数据库，将整合来自多个LINCS实验室的分析结果。最终产品将是一个高信息含量的多重质谱分析，称为“加速蛋白质信号签名”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，以便以协作的方式在多个蛋白质组学实验室中实际实施。

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.