INTERACT: Interactome Network Targeting via Enzyme Reactivity and Activity-based Chemoproteomic Tools

Abstract

This chapter details an activity-based chemoproteomic methodology, INTERACT (Interactome Network Targeting via Enzyme Reactivity and Activity-based Chemoproteomic Tools), for the functional interrogation of host-pathogen interactions (interactome) in a physiologically relevant context. The exemplar protocol utilises a small, cell-permeable fluorophosphonate probe to covalently label active serine hydrolases simultaneously within Leishmania mexicana parasites and their murine macrophage host cells during active infection. Subsequent biorthogonal click chemistry, affinity enrichment, and quantitative mass spectrometry using Tandem Mass Tags (TMT) enable the identification and relative quantification of functionally active enzymes across the interactome. This method facilitates the delineation of dynamic changes in enzyme activity during the infection process, yielding rich insight into host-pathogen biochemical networks, including the identification of pathogen virulence factors and host responses. Therefore, INTERACT delivers a robust and quantitative workflow to probe enzymatic activity across two species in a single experiment under native infection conditions, without the need for genetic manipulation, providing an invaluable platform for dissecting pathogenesis and uncovering novel therapeutic targets in infectious diseases.