Methods to elucidate quantitative phosphorylation dynamics in the IGF-1 signaling pathway

项目总结 细胞信号通路中与磷酸化相关的扰动是许多人类疾病的根源。 蛋白质的磷酸化不能用DNA或RNA测序观察到，这意味着它非常 重要的是开发可重复性和准确定量的蛋白质组学技术和方法 磷酸化事件。串联质谱仪目前提供了一个很好的平台来深入分类 与疾病相关的磷酸化部位。然而，准确的重复性和量化是 受阻于几个收购权衡，以提供更高的低-丰富的识别率 分子。我提出了一种在磷酸化实验中提高定量重复性的方法， 特别是关于位置磷酸化异构体。我打算构建计算工具， 在蛋白质组工作流程中分离多肽鉴定和位点定位的任务。我会在现场表演 使用利用由多个扫描组成的整个碎片离子分布计算的几个度量进行定位， 而不是目前只使用一次扫描来定位磷酸化位点的方法。这应该是 显著提高了准确性和重复性。我将应用这项技术来监测磷酸化事件 在IGF-1信号通路中帮助阐明人类细胞系对各种刺激的特异性 这条路径的成员之间的网络特征。该项目也将极大地惠及信令 社区通过提供工具和方法来更可靠地发现 磷酸蛋白质组。

PROJECT SUMMARY Phosphorylation related perturbations in cellular signaling pathways are at the root of many human diseases. Protein phosphorylation cannot be observed using DNA or RNA sequencing, which means it is extremely important to develop proteomic technologies and methods that reproducibly and accurately quantify phosphorylation events. Tandem mass spectrometry currently provides an excellent platform to deeply catalog the sites of phosphorylation with respect to disease. However, accurate reproducibility and quantitation are hampered by several acquisition tradeoffs made to provide higher identification rates of low-abundant molecules. I propose an approach to improve quantitative reproducibility in phosphorylation experiments, specifically with regard to positional phosphorylation isomers. I intend to build computational tools that separate the tasks of peptide identification and site localization in proteomic workflows. I will perform site localization using several metrics calculated with entire fragment ion profiles made up of multiple scans, instead of current methods that only use a single scan to localize phosphorylation sites. This should significantly improve accuracy and reproducibility. I will apply this technique to monitor phosphorylation events in the IGF-1 signaling pathway of human cell lines with respect to various stimuli to help elucidate specific networking characteristics between members of that pathway. This project will also greatly benefit the signaling community by providing tools and methods to more reliably discover quantitative changes in the phosphoproteome.