Adaptation of the Protocol for the Isolation of Biotinylated Protein Complexes for Drosophila melanogaster Tissues

Proximity biotinylation, which utilizes various biotin ligating enzymes (BioID, TurboID, etc.), is widely used as a powerful tool for identifying novel protein-protein interactions. However, this method has a significant limitation: the use of streptavidin on beads for enriching biotinylated proteins often results in a high background of peptides from streptavidin itself, which interferes with identification by peptide mass fingerprinting. This limitation makes it practically impossible to study samples containing a small amount of material, such as individual insect tissues. In this study, we compared different precipitation and elution conditions for the purification of biotinylated proteins from protein extracts of <i>Drosophila melanogaster</i> S2 cells. We found that biotinylated proteins can be purified using anti-biotin antibodies, although with lower efficiency than streptavidin-based resin. We also demonstrated that protease-resistant streptavidin (prS), previously tested in mammalian cells, can be used effectively to purify biotinylated proteins from tissues of <i>D. melanogaster</i>. In our experiments, prS showed precipitation efficiency comparable to regular streptavidin but generated a lower background in peptide fingerprinting. To further demonstrate the applicability of prS for studying protein-protein interactions in <i>D. melanogaster</i> tissues, we carried out experiments to identify interaction partners of the ecdysone receptor (EcR) in <i>D. melanogaster</i> ovarian tissue using TurboID-based proximity biotinylation. As a result, EcR was found to interact with both previously described and novel protein partners in <i>Drosophila</i> ovaries.