A systems-level approach to decipher the protein interactome.

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.

Grant Number: 4DP2GM146246-02
NIH Institute/Center: NIH
Principal Investigator: Keriann Backus