Protein fishing using magnetic nanobeads containing calmodulin site-specifically immobilized via an azido group

We developed a novel method for capturing proteins that interact with a target protein. This method utilizes a protein containing a site-specifically incorporated 3-azidotyrosine (N_3-Y) and FG beads for immobilization of the protein via an azido group. Using calmodulin (CaM) as the target protein, we introduced N_3-Y at position 72 and conjugated it to FG beads by copperfree click chemistry. From the Ca²⁺/CaM-binding proteins captured from mouse brain cell lysate and analysis by mass spectrometry, we identified six proteins: alpha-enolase (ENOA), glucose-6-phosphate isomerase (GPI), annexin A5 (ANXA5), malate dehydrogenase 1 (MDH1), glyceraldehyde-3- phosphate dehydrogenase and the well-known CaM-binding protein phosphoglycerate kinase 1 (PGK1). The presence of photo-crosslinking products via N_3-Y for all the captured proteins except GPI indicated that they bound directly to CaM. In this study, ENOA, ANXA5 and MDH1 were identified as novel CaM-binding proteins, and PGK1 was bound to Ca²⁺/CaM and also Ca ²⁺-free CaM. This method should prove useful for capturing novel interacting proteins and serve as a useful tool for proteomic analyses.