title: Protein fishing using magnetic nanobeads containing calmodulin site-specifically immobilized via an azido group.
abstract: 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 (N3-Y) and FG beads for immobilization of the protein via an azido group. Using calmodulin (CaM) as the target protein, we introduced N3-Y at position 72 and conjugated it to FG beads by copper-free click chemistry. From the Ca(2+)/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 N3-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 Ca(2+)/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.
authors: Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors: Akiyoshi
authors: A
authors: Ikeda-Boku
authors: Keisuke
authors: K
authors: Kondo
authors: Satoshi
authors: S
authors: Ohno
authors: Erika
authors: E
authors: Yoshida
authors: Takashi
authors: T
authors: Yokogawa
authors: Nobuhiro
authors: N
authors: Hayashi
authors: Kazuya
authors: K
authors: Nishikawa
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D002147:Calmodulin / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry*; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
chemical_list: D002147:Calmodulin; D055767:Immobilized Proteins; D058185:Magnetite Nanoparticles; D009419:Nerve Tissue Proteins
keywords: 3-azidotyrosine; FG beads; calmodulin; photo-crosslinking; protein–protein interaction