Coupling Affinity Purification with Proximity Labeling in Arabidopsis: The APEAL Approach.

Capturing interactions in transient protein assemblies is a challenging task. We introduce an approach named APEAL (Tandemly Coupled Affinity Purification with Proximity-dEpendent LigAtion) for determining both transient and stable protein assemblies in Arabidopsis and other plants. APEAL exploits TurboID, a biotin ligase variant that operates efficiently at "plant-friendly" temperatures. The APEAL method begins by enriching strong or long-lived protein-protein interactions through an affinity purification step. It then captures weaker and more transient interactions through a proximity-dependent ligation step. At both stages, samples can be analyzed using immunoblotting or mass spectrometry to extract relevant data. This chapter details the APEAL procedure from construct design and pilot experiments to condition testing, proteomic sample collection, and mass spectrometry data analyses.