Biosensor design for detection and analysis of complex signal patterns in cancer

描述（由申请人提供）：以相关方式研究生物系统的异常复杂性需要多维度和高度具体的分析。最终，我想建立一个化学生物学研究项目，使用合成蛋白质化学和系统生物学方法，以简单的方式解决研究复杂性的挑战。我计划使用化学蛋白质合成作为一种组合策略来产生具有足够特征的分子生物传感器，以定义与乳腺癌相关的激酶信号通路中的差异和特征，并且还允许结合化学技巧来改善信号对噪声的影响并简化分析。这些生物传感器将在检测、监测和设计癌症相关途径的治疗方面具有临床应用价值，并将提供有关相关激酶活性与癌症疾病状态相关性的独特信息，具有高信号噪声、分辨率和特异性。具体目标是：建立和表征人工靶向激酶底物蛋白的初始文库；使用这些人工底物在体内检测癌症相关的激酶活性；并在系统级方法中使用体内磷酸化信息来定义与癌症相关的多底物“特征”，以作为疾病状态的生物传感器。为了制造这些生物传感器，将使用固相肽合成技术合成具有不同特定功能的小肽单元，并通过天然化学连接相互连接，制成小的（100-150 aa）人工蛋白质。这些蛋白质将由模块组成，用于指导其细胞内化和定位，增强特定蛋白质-蛋白质相互作用，通过荧光报告其位置，并作为特定信号通路的激酶底物。激酶底物部分将被选择性释放，并包含一个亲和标签，用于快速纯化该部分，并使用MALDI-TOF质谱作为激酶活性的报告因子进行分析。在这个项目中，人工蛋白质生物传感器将被设计用来帮助我们可视化酪氨酸激酶异常活性在乳腺癌等疾病中的复杂模式。在许多乳腺癌中，这些酪氨酸激酶非常丰富且过度活跃，破坏了正常的细胞过程，使生长不受控制。了解它们的活动是如何相互关联的，以及它们模式变化的时机，将有助于我们更好地诊断和治疗表现出这些特征复杂性的疾病。

DESCRIPTION (provided by applicant): To study the extraordinary complexity of biological systems in a relevant way requires both multidimensional and highly specific analyses. Ultimately, I want to build a chemical biology research program using synthetic protein chemistry and a systems biology approach to address the challenges of studying complexity in a simple way. I plan to use chemical protein synthesis as a combinatorial strategy to generate molecular biosensors with enough features to define differences and signatures in kinase signaling pathways related to breast cancer, and that also allow for the incorporation of chemical tricks to improve signal to noise and ease analysis. The biosensors will have clinical applications in detecting, monitoring and designing treatment for cancer-related pathways, and will provide unique information about the relevance of interrelated kinase activities to cancer disease states with high signal to noise, resolution and specificity. The specific aims are to: build and characterize an initial library of artificial, targeted kinase substrate proteins; use these artificial substrates for in vivo detection of cancer-related kinase activities; and use the in vivo phosphorylation information in a systems-level approach to define cancer-related multi-substrate 'signatures' to serve as biosensors for disease states. To generate these biosensors, small peptide units with different specific functions will be synthesized using solid-phase peptide synthesis and linked to each other using native chemical ligation to make small (100-150 aa) artificial proteins. These proteins will consist of modules for directing their cellular internalization and localization, enhancing specific protein-protein interactions, reporting their location via fluorescence and acting as kinase substrates for particular signaling pathways. The kinase substrate portion will be selectively releasable, and contain an affinity tag for rapid purification of this segment and analysis using MALDI-TOF mass spectrometry as a reporter for kinase activity. In this project, artificial protein biosensors will be designed to help us visualize the complicated patterns of abnormal tyrosine kinase activity in diseases like breast cancer. In many breast cancers, these tyrosine kinases are highly abundant and overactive, disrupting the normal cellular processes that keep growth from getting out of control. Understanding how their activities interrelate and the timing of changes in their patterns will help us better diagnose and treat diseases that show these characteristic complexities.