Abstract Protein interactions, such as protein-protein, protein-nucleic acid, protein-metabolite, and protein-xenobiotic, play a critical role in defining the uniqueness and complexity of biological organisms. Understanding where and when interactions occur is an essential step to functionally characterize the interactome. However, and despite remarkable advances in computational and proteomic technologies, it remains surprisingly difficult to precisely pinpoint contact sites cell-wide in a high-throughput manner. Here we present a new approach, termed Fast Photochemical Oxidation and Capture by Suzuki (FPICS), to map protein interaction sites at high resolution. The key innovation of our method, which represents an unprecedented technical advance, is the use of a single halogen atom as both a photoactivatable molecular 'calling card,' to indicate where interactions occur, and a capture handle, for mass spectrometry-based proteomic detection of each interaction site. With FPICS, halogen substituents are first transferred, using excimer laser irradiation, from halogenated small molecules (e.g. drugs or natural products) or halogenated biomolecules (e.g. proteins, lipids, glycans, oligonucleotides, or metabolites) to interacting proteins. Labeled sites are then captured and identified using bioorthogonal Suzuki–Miyaura cross- coupling chemoproteomic methodology pioneered by our group. FPICS is groundbreaking because it eliminates challenges associated with deconvolving the spectra of crosslinked peptides and the frequent and unwanted fragmentation of large biomolecules. Showcasing the method's wide-ranging applications, here we will apply FPICS map the protein interaction sites for small molecules, lipids, and nucleic acids, aiming to identify new functional and therapeutically relevant binding sites proteome-wide. Taken together, this study will yield a systems-level portrait of the protein interactome, which will lay the foundation for an improved global understanding of the functional significance of the millions of interactions occurring within every cell. The impact of our methods will be wide ranging, spanning the fields of chemical biology, analytical chemistry, and systems biology.

Project Narrative Understanding how proteins work and the interactions that they participate in is the bedrock of functional biology and drug development. Most protein interactions remain uncharacterized. By developing an entirely new approach to map protein contact sites, this proposal will revolutionize our understanding of these interactions and will provide a roadmap for future studies aimed at functionally charactering and pharmacologically manipulating the protein interactome.