Accelerated Protein Signaling Signatures

LINCS计划旨在推导出细胞扰动产生的分子特征。蜂窝 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，是互补的转录谱。该提案详细介绍了发展高 用于查询丝氨酸和苏氨酸信号传导基于信息含量多路质谱的测定 途径。一种还原主义的方法，即多个磷酸化位点的自然相关性， 不同的蜂窝条件被阐明，将被用来创造效率，在代表蜂窝 状态为了实现这一点，在不同条件下， 条件将使用质谱法（MS）获得。从这些数据来看， 将提取代表性的磷酸肽-简化的代表集-其水平和 化学计量将用于捕获响应于扰动的特征。随后，必要的 将采购试剂，以便设计靶向质谱分析（多反应 监测或MRM-MS），以在多重（~100重）中定量简化代表集中的肽 时尚.变异性、重现性、检测限和定量限以及最终检测成本将在 测定了还将展示该检测的多实验室实施。与此同时， 努力，建立开放获取蛋白质组学软件Skyline将被扩展，以提供一个标准 容纳重要测定参数的方法，处理从简化表示集获得的数据 MRM-MS测定，并整合QA/QC指标以确定测定性能。天际线也将是 适应允许MRM-MS方法和数据的实验室间交换，我们将开发一个公共的 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 称为“加速蛋白质信号转导标签”的信息内容多重MS测定和 信息基础设施，以支持包含所有必要信息的社区资源， 我们提供了一种工具，以协作方式在多个蛋白质组学实验室中实际实施。

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.