Technology for evaluating drug-binding responses to small-molecule perturbation

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PROJECT SUMMARY | ABSTRACT Proteins act as the effector molecules of cells – carrying out most of the structural, regulatory, and enzymatic functions. Proteins themselves are often regulated through direct interaction with ligands, including metals, lipids, other proteins, and drugs. These protein-ligand interactions are fundamental to diverse biological processes. Yet, technologies to explore these interactions are limited in terms of both throughput and their ability to scale. The limits of these technologies are in part highlighted be the fact that for nearly 30 years, proteomics and genomics technologies research have been unable to fully characterize the functions of the 20,000 protein coding genes in human cells. To address this, we propose to build a cornerstone technology suite for high-throughput, proteome-wide protein-ligand interaction profiling. In this work we will demonstrate the development and implementation in a focused way to highlight the potential of this technology to bring robust quantitative approaches to study ligand binding at scale. Our technological innovations center on using high-throughput methods to detect protein- ligand interactions across the entire proteome in a single analysis. To do this, we will measure the change in thermal stability of proteins induced by binding to a ligand. We measure this thermal stability as a relative difference in protein abundance using sample multiplexing based on tandem mass tags (TMT). Sample multiplexing enables quantitation of up to 18 samples’ proteomes simultaneously. Sample multiplexing with TMT increases sample throughput, reduces missing values across samples, and enables complex experimental designs – e.g., time courses, dose dependency, and knockout-rescue experiments. Over the course of the proposed work, we will build new proteomics technologies to harness the benefits of proteome-wide thermal stability assays and TMT quantitation to characterize protein-ligand interactions. The combination of (1) intelligent mass spectrometric data acquisition, (2) proteome thermal stability profiling, and (3) sample multiplexing will enable us to decipher the complex interplay between proteins and ligands across the proteome. With an eye towards translational research, we will focus at first on small-molecule drugs as ligands as we can acquire diverse libraries with known primary protein targets. These data and methods will be used to reveal the functional and secondary effects of ligand perturbation of the proteome by leveraging matched whole proteome and gene expression profiles to determine to what extent specific drug-protein- engagement drives cellular responses.