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

项目摘要 酪氨酸磷酸化是一种翻译后修饰的fi阳离子，可以调节蛋白质的功能和相互作用。 特兹。酪氨酸磷酸化在癌症中经常变得失调，因此，了解 蛋白质功能和生化网络中的磷酸化将对确定治疗方案至关重要 癌症的干预措施。不幸的是，许多酪氨酸的磷酸化被称为“暗磷酸-- 因为我们要么无法检测到它，要么我们不了解它在调节蛋白质和 网络。由于测试和测量pTyr的工具的局限性，存在着黑暗的磷酸蛋白质组 在蛋白质和网络中。在之前的工作中，我们开发了一种合成工具包，它具有优异的PHY产量- 与现有方法相比，感兴趣蛋白质上生物学上相关的酪氨酸磷酸化。目标是 这项工作的目的是：1)强化这项技术，以提高重复性和fl灵活性；2)推广这项技术-- OGY以目前成本的一小部分生产试剂，用于定量测量和监测 质谱学研究中的磷酸酪氨酸位点fl与癌症进展和治疗的关系 回应。

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.