Abstract
Protein-protein interactions (PPIs) are fundamental regulators of cellular function and disease. Systematic mapping of the interactome is essential for identifying therapeutic targets and advancing drug design, a pursuit that has driven significant innovation to capture the spatiotemporal regulation of PPIs in vivo. This review summarizes this methodological revolution. We outline foundational, first-generation techniques-yeast two-hybrid and co-immunoprecipitation-which established frameworks for binary interaction mapping and static network generation, especially when integrated with mass spectrometry. The discussion then pivots to second-generation methods, including proximity-dependent labeling and advanced imaging, which enable the capture of PPIs within their native, dynamic cellular contexts. We provide a comparative analysis of these techniques, detailing their principles, strengths, and limitations. The review concludes with a practical framework for method selection and a perspective on emerging frontiers-such as spatial proteomics and single-cell interactomics-that are poised to further decode the evolving interactome. This concise overview serves as a strategic guide for specialists adopting new techniques and a broader audience integrating network-level data into their research.