title: Tandem photoaffinity labeling of a target protein using a linker with biotin, alkyne and benzophenone groups and a bioactive small molecule with an azide group.
abstract: A novel linker containing biotin, alkyne and benzophenone groups (1) was synthesized to identify target proteins using a small molecule probe. This small molecule probe contains an azide group (azide probe) that reacts with an alkyne in 1 via an azide-alkyne Huisgen cycloaddition. Cross-linking of benzophenone to the target protein formed a covalently bound complex consisting of the azide probe and the target protein via 1. The biotin was utilized via biotin-avidin binding to identify the cross-linked complex. To evaluate the effectiveness of 1, it was applied in a model system using an allene oxide synthase (AOS) from the model moss Physcomitrella patens (PpAOS1) and an AOS inhibitor that contained azide group (3). The cross-linked complex consisting of PpAOS1, 1 and 3 was resolved via SDS-PAGE and visualized using a chemiluminescent system. The method that was developed in this study enables the effective identification of target proteins.
authors.affiliation: a Research Faculty of Agriculture, Division of Fundamental Agriscience Research , Hokkaido University , Sapporo , Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Anabuki
authors.affiliation: a Research Faculty of Agriculture, Division of Fundamental Agriscience Research , Hokkaido University , Sapporo , Japan.
authors.forename: Miu
authors.initials: M
authors.lastname: Tsukahara
authors.affiliation: a Research Faculty of Agriculture, Division of Fundamental Agriscience Research , Hokkaido University , Sapporo , Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Matsuura
authors.affiliation: a Research Faculty of Agriculture, Division of Fundamental Agriscience Research , Hokkaido University , Sapporo , Japan.
authors.forename: Kosaku
authors.initials: K
authors.lastname: Takahashi
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D001577:Benzophenones / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D019920:Photoaffinity Labels; D011506:Proteins / Q000737:chemistry*; D066244:Proton Magnetic Resonance Spectroscopy
chemical_list: D000480:Alkynes; D001386:Azides; D001577:Benzophenones; D019920:Photoaffinity Labels; D011506:Proteins; D001710:Biotin
keywords: Huisgen cycloaddition; Western blotting; linker; photoaffinity labeling; target protein