Refined affinity purification protocol for CAMs using minimal mouse brain material

Abstract

Background Cell adhesion molecules (CAMs) are membrane-bound proteins that mediate cell-cell interactions through trans-cellular protein complexes. In the context of the neuronal synapse, studies of CAMs have revealed their roles from neuronal recognition and neuronal wiring to synaptic plasticity. CAMs form macromolecular complexes via cis- and trans-interactions; however, identifying the specific proteins in these assemblies is challenging. Their interactions are dynamic and transient, making them difficult to capture, and their hydrophobic transmembrane domains complicate extraction from biological samples. New method Here, we present a protocol to pulldown interacting partners of a Teneurin-3-GFP bait protein, as a representative CAM, from minimal mouse brain lysate. Comparison with existing methods Affinity purification of a bait protein from a biological sample, followed by mass spectrometry to identify captured prey proteins is a widely used, unbiased approach, though it usually requires large amounts of material. We show that our refined approach detects known Teneurin interactants while substantially reducing the animal tissue required. We further compared detergents used for lysate preparation and found that the total of CAM species enriched in Teneurin-3 samples relative to control varied considerably. Finally, we evaluated different normalization workflows to aid dataset interpretation. Conclusion This protocol provides an accessible approach for studying CAM interactions with limited animal tissue, enabling refined insights into the complex protein networks underlying synaptic connectivity.