EAGER: Comprehensive monitoring of protein interaction networks

基因组编码了数以千计的蛋白质的信息，这些蛋白质构成了所有生物体中细胞机械的基石。蛋白质通常不是孤立地发挥作用，而是在多组分的复合体中发挥作用，因此最重要的细胞功能是由蛋白质之间的基本相互作用决定的。这些蛋白质相互作用网络是动态的，相互作用的变化是细胞和组织功能的生理和病理变化的主要原因。因此，必须拥有通用且易于实现的工具，使科学家能够测量蛋白质相互作用的动力学。该项目旨在开发这样一种工具，使研究人员能够在多种条件下同时绘制多达数百种蛋白质的动态蛋白质相互作用图。这一新方法将解决生物学中的许多关键问题，例如哪些交互作用变化驱动发育决定，哪些交互作用变化是由于导致病理结果的突变而发生的。此外，该项目在推进生物技术方面具有很高的潜力，例如，通过使系统地筛选候选药物具有特定破坏驱动疾病状态的蛋白质相互作用的能力。该项目还为研究生和本科生提供了跨学科的研究培训机会。该项目将采用一种新的基于抗体和测序的策略，以绘制蛋白质相互作用及其变化的图谱。该战略有两个基本组成部分。首先，抗体由一段短的DNA序列进行独特的“条形码”编码，因此每一种抗体--以此类推，其目标蛋白--都可以通过下一代测序轻松识别。第二，组合条形码策略，通过拆分和汇集程序，将独特的组合DNA序列添加到条形码抗体中。只有共同结合到同一蛋白质复合体的抗体才会得到相同的组合条形码，而蛋白质之间的相互作用可以通过对这些条形码进行测序和匹配来重建。总而言之，该项目旨在提供一种简便的方法，在高通量下直接研究蛋白质相互作用动力学，但同时在单一复杂的分辨率下。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The genome encodes information for thousands of proteins, which form the building blocks of cellular machinery in all organisms. Proteins normally do not function in isolation, but rather in multi-component complexes, such that most vital cellular functions are governed by essential interactions between proteins. These protein interaction networks are dynamic, and changes in the interactions are the major cause of physiological and pathological changes in cellular and organismal function. Thus, it is essential to have universal and easily implementable tools that allow scientists to measure the dynamics of protein interactions. This project aims to develop such a tool, which will allow researchers to simultaneously map dynamic protein interactions for up to hundreds of proteins under many conditions. This novel approach will address many key issues in biology, such as which interaction changes drive developmental decisions or occur due to mutations that lead to pathological outcomes. Moreover, the project has high potential to advance biotechnology, for example, by making it possible to systematically screen drug candidates for the ability to specifically disrupt protein interactions that drive a disease state. The project also provides cross-disciplinary research training opportunities for graduate and undergraduate students.The project will employ a new antibody and sequencing-based strategy in order to map protein interactions and their changes. There are two fundamental components to the strategy. First, antibodies are uniquely “barcoded” by a short DNA sequence so that each antibody – and by extension, its target protein – can be easily identified by next-generation sequencing. Second, a combinatorial barcoding strategy, via a spilt and pool procedure, adds unique combinatorial DNA sequences to the barcoded antibodies. Only antibodies co-bound to the same protein complex will get the same combinatorial barcode and protein interactions can be reconstructed by sequencing and matching these barcodes. In summary, this project aims to provide a facile approach to directly study protein interaction dynamics at high throughput, but at the same time at single complex resolution.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.