EAGER: Comprehensive monitoring of protein interaction networks

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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.