Antibody-Mediated Protein A-APEX2 Labeling (AMAPEX) for Proximity Proteome Exploration.

Exploring protein interactions is essential for understanding cellular biological processes. Traditional methods for studying protein-protein interactions often face limitations in capturing transient or low-affinity interactions due to harsh lysis conditions or antibody specificity challenges. Proximity labeling has emerged as a robust technique that utilizes engineered enzymes to covalently tag proximal proteins, preserving their interactions in vivo. This chapter introduces an advanced method called antibody-mediated protein A-APEX2 labeling (AMAPEX), which enhances the established APEX2 system. AMAPEX utilizes specific antibodies to tether a modified protein to a protein A-APEX2 fusion, enabling targeted biotin labeling of proximal proteins. These labeled proteins are subsequently isolated using streptavidin beads and analyzed by mass spectrometry. Validation of the method is demonstrated by profiling histone modification proxeomes, demonstrating its accuracy in identifying associated cellular components. AMAPEX represents a significant advancement in mapping protein interactions, particularly in the context of posttranslational modifications, making it a valuable tool for biological research and therapeutic development.