Methods for the Investigation of Protein–Ligands Interactions

Biomolecular interactions involving proteins, nucleic acids, and small molecules constitute the molecular foundation of cellular regulation, signaling, and therapeutic intervention. Advances in mass spectrometry–based proteomics have enabled the systematic characterization of these interactions at unprecedented depth, sensitivity, and structural resolution. This chapter provides a comprehensive overview of state-of-the-art proteomics methodologies developed to investigate protein–protein, protein–nucleic acid, and protein–drug interactions, with particular emphasis on experimental design, sample preparation, and data quality control. Targeted and untargeted strategies are discussed, including affinity purification–mass spectrometry, proximity-dependent labeling, cross-linking mass spectrometry, blue native electrophoresis, and size-exclusion chromatography–mass spectrometry for protein–protein interactions; affinity capture, EMSA-MS, chromatin immunoprecipitation–mass spectrometry, CRISPR-based locus-specific enrichment, and CLIP-based approaches for protein–nucleic acid complexes; and chemoproteomics, thermal proteome profiling, and label-free structural proteomics for protein–drug interaction analysis. The chapter further highlights recent technological innovations, computational tools, and integrative multi-omics strategies that enhance interaction mapping across biological scales. By critically evaluating the strengths, limitations, and appropriate applications of each methodology, this work aims to provide practical guidance for researchers seeking to design robust interactomics experiments and to interpret complex molecular networks in both basic and translational research contexts.