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

项目摘要 细胞信号传导途径中的磷酸化相关扰动是许多人类疾病的根源。 使用DNA或RNA测序不能观察到蛋白质磷酸化，这意味着它是非常重要的。 重要的是开发蛋白质组学技术和方法， 磷酸化事件。串联质谱目前提供了一个很好的平台， 与疾病相关的磷酸化位点。然而，准确的重现性和定量是 阻碍了几个收购权衡，以提供更高的识别率低丰度 分子。我提出了一种方法，以提高定量重复性磷酸化实验， 特别是关于位置磷酸化异构体。我打算构建计算工具， 在蛋白质组学工作流程中分离肽鉴定和位点定位的任务。我将执行网站 使用利用由多次扫描组成的整个碎片离子轮廓计算的若干度量进行定位， 而不是仅使用单次扫描来定位磷酸化位点的现有方法。这应该 显著提高了准确性和再现性。我将应用这项技术来监测磷酸化事件 在IGF-1信号通路的人类细胞系相对于各种刺激，以帮助阐明具体的 这条道路上的成员之间的网络特征。这个项目也将大大有利于信号 通过提供工具和方法，更可靠地发现 磷酸化蛋白质组

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.