Beyond Proximity: Reactive Intermediates for Mechanistic Interactome Mapping

Interactome studies have transformed biology by showing that cellular function emerges from dynamic networks of protein-protein interactions rather than from isolated proteins acting alone. Yet the field faces a central unsolved problem: most current methods capture spatial proximity more readily than they resolve direct, biologically meaningful interaction states. The next advances will come not simply from additional labeling platforms, but from improved chemistries and reactive intermediates that more precisely match each platform to the biological claim it is intended to support. Biotinoyl-5′-AMP, radicals, carbenes, nitrogen-centered intermediates, electrophiles, and covalent crosslinks each impose a distinct balance of capture breadth, residue selectivity, spatiotemporal control, and mechanistic interpretability. These platforms also differ in experimental scope: bait-localized enzyme and photocatalyst systems usually interrogate one preselected protein or microenvironment at a time, whereas crosslinking-MS can sample many interaction surfaces in parallel without functionalizing a specific protein. This Perspective argues that reactive-intermediate design provides a unifying chemical logic for the next generation of interactome mapping. By integrating organic chemistry, photochemistry, inorganic chemistry, biocatalysis, proteomics, and computation, the field can move beyond descriptive proximity maps toward dynamic and mechanistically informative views of protein interaction networks in cells, tissues, disease states, and ultimately living organisms.