Protocol to study secretome interactions using extracellular proximity labeling.

Abstract

Biotin ligase-based proximity ligation is a widely used, highly effective technique for the study of in vivo protein-protein interactions. However, there are few reports and little consensus on the most effective methods for studying the proximal interactomes of secreted factors. Here, we present a protocol for studying extracellular proximal interactomes using an adaptation of TurboID/BioID2-based proximity ligation. We describe steps for cell preparation, sample collection, and initial processing. We then detail procedures for biotinylated protein enrichment, on-bead digestion, and post-pull-down processing. For complete details on the use and execution of this protocol, please refer to Peeney et al.¹.