Mapping Dysfunctional Protein-Protein Interactions in Disease.

Protein-protein interaction (PPI) networks are dynamically remodeled in disease, yet most systems biology approaches focus on changes in protein abundance, overlooking critical interaction-level dysfunction. Here, we present a robust, chemoproteomic method-dysfunctional Protein-Protein Interactome (dfPPI)-that enables high-throughput, systematic, disease-contextual mapping of PPI network dysfunctions in cells and primary human tissue. This method integrates chemical biology probes that selectively capture epichaperome-based interactome assemblies with label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and network-based computational analysis, to uncover the rewiring of protein networks not apparent from transcriptomic or proteomic data alone. The dfPPI platform can be applied across disease states, species, and tissues to identify actionable nodes of dysfunction and enable high-resolution, systems-level insights into disease progression. In this protocol, we demonstrate step-by-step procedures for sample preparation, chemical probe treatment, affinity enrichment, label-free LC-MS/MS analysis, and bioinformatics workflows used to generate and interpret dfPPI datasets. This article aims to promote reproducibility and accessibility of this approach, supporting its adoption by the broader systems biology and translational research communities.