Accelerated Protein Signaling Signatures

Lincs计划寻求从细胞扰动中获得分子签名。蜂窝 通过调节蛋白质磷酸化来传递信号是细胞反应的重要组成部分 对刺激，并且是转录图谱的补充。这项建议详细说明了一种高 基于信息内容多重质谱学的丝氨酸和苏氨酸信号查询分析 小路。一种简化论的方法，在这种方法下，多个磷酸化位点的自然相关性 不同的细胞条件被阐明，将被用于在代表细胞的过程中创造效率 州政府。为了实现这一点，在不同的条件下，适量的全球磷蛋白质组定量图谱 条件将使用质谱仪(MS)获得。从这些数据中，有限数量的 将提取具有代表性的磷酸肽-简化的表示集-其水平和 化学计量学将用于捕捉响应扰动的签名。随后，必要的 将采购试剂以允许设计靶向质谱分析(多重反应 监测，或MRM-MS)，以对多路(~100-plex)中简化表示集中的多肽进行量化 时尚。可变性、重复性、检测和定量的限度，以及最终的化验成本 量过了。还将演示该分析的多个实验室实现。与此平行的是 经过努力，已建立的开放获取蛋白质组学软件Skyline将得到扩展，提供一个标准 存储重要的分析参数、处理从简化表示集获取的数据的装置 MRM-MS分析，并整合QA/QC指标以确定分析性能。天际线也将是 适应于允许实验室间交换MRM-MS方法和数据，我们将开发一种公共的 将整合来自多个Lincs实验室的检测结果的数据库。最终的产品将是一个很高的 被称为“加速蛋白信号特征”的信息内容多重质谱分析 支持包含所有必要信息的社区资源的信息学基础设施 以协作的方式在多个蛋白质组学实验室中实际实施的工具。

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.