Accelerated Protein Signaling Signatures

描述(由申请人提供)：Lincs计划寻求从细胞扰动中获得分子签名。通过调节蛋白质磷酸化的细胞信号是细胞对刺激的反应的重要组成部分，是转录图谱的补充。这项建议详细说明了一种基于高信息含量的多重质谱学分析方法的开发，以查询丝氨酸和苏氨酸信号通路。一种简化论的方法，即阐明不同细胞条件下多个磷酸化位点的自然相关性，将被用来创造代表细胞状态的效率。为了实现这一点，将使用质谱学(MS)在不同条件下获得适度数量的全球磷蛋白质组定量图谱。从这些数据中，将提取有限数量的具有代表性的磷酸肽-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，将采购必要的试剂，以允许设计靶向质谱分析(多反应监测，或MRM-MS)，以多重(~100-plex)方式定量简化表示集的多肽。将测量可变性、重复性、检测和定量的限度以及最终的检测成本。还将演示该化验的多个实验室实施。在这些努力的同时，已建立的开放获取蛋白质组学软件Skyline将得到扩展，以提供存储重要分析参数、处理从简化表示集MRM-MS分析中获得的数据以及整合QA/QC指标以确定分析性能的标准方法。天际线还将被改造，以允许实验室之间交换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。最终产品将是一种被称为“加速蛋白质信号特征”的高信息含量多重MS分析，以及支持社区资源的信息基础设施，该社区资源包含以合作方式在多个蛋白质组实验室中实际实施所需的所有信息和工具。 与公共卫生相关：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，而时间和成本只是类似方法的一小部分。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.