A synthetic toolkit for the recombinant production of tyrosine phosphorylated proteins and peptides

项目摘要 酪氨酸磷酸化（pTyr）是一种翻译后修饰，可以调节蛋白质功能和相互作用。 选择。酪氨酸磷酸化通常在癌症中变得失调，因此，了解酪氨酸磷酸化的作用， 蛋白质功能和生化网络内的磷酸化将是确定治疗性药物的最重要因素。 干预癌症。不幸的是，许多酪氨酸磷酸化被称为“暗磷酸化”， teome”，因为我们要么无法检测到它，要么我们不知道它在调节蛋白质中的作用， 网络.暗磷酸蛋白质组的存在是由于测试和测量pTyr的工具的局限性 在蛋白质和网络中。在以前的工作中，我们开发了一种合成工具包，具有上级产量的phys- 与现有的方法相比，本发明的方法在感兴趣的蛋白质上具有生物学相关的酪氨酸磷酸化。目标 这项工作的目的是：1）强化这项技术，以提高再现性和可重复性，2）扩展这项技术， ogy以目前成本的一小部分生产用于定量测量和监测的试剂 在基于质谱的工作中，磷酸酪氨酸位点与癌症进展和治疗相关 反应

Project Summary Tyrosine phosphorylation (pTyr) is a post-translational modification that can regulate protein function and interac- tions. Tyrosine phosphorylation often becomes dysregulated in cancer and therefore, understanding the effect of phosphorylation on protein function and within biochemical networks will be paramount to identifying therapeutic interventions in cancer. Unfortunately, much of tyrosine phosphorylation is referred to as the “dark phosphopro- teome”, because we either cannot detect it or we do not understand what its role is in regulating proteins and networks. The dark phosphoproteome exists as a result of limitations in tools for testing and measuring pTyr within proteins and networks. In previous work, we developed a synthetic toolkit that has superior yields of phys- iologically relevant tyrosine phosphorylation on proteins of interest, compared to existing approaches. The goal of this work is to: 1) harden this technology for increased reproducibility and flexibility and 2) extend this technol- ogy to produce reagents, at a fraction of their current cost, for use in quantitative measurement and monitoring of phosphotyrosine sites in mass spectrometry-based workflows relevant to cancer progression and treatment response.