Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Development of gold nanoparticle-based clickable photoaffinity probes for target protein identification (#734)

Kanna Mori 1 , Kaori Sakurai 1
  1. Tokyo University of Agriculture and Technology, Graduate School of Engineering, Koganei City, TOKYO, Japan

Target protein identification is the first critical step for the elucidation of the mechanism of action of bioactive small molecules and in the field of drug discovery.  Photoaffinity labeling has been useful for target identification studies because it enables covalent crosslinking of target proteins in live cells.  However, its routine application is hampered by the need to optimize probe design, high background labeling, and a multistep scheme.  We have previously developed gold nanoparticle (AuNP)-based photoaffinity labeling probes and successfully improved probe synthesis and labeling efficiency by taking advantage of the multivalent effect and the unique property of AuNP.  In this study, we devised new clickable photoaffinity probes based on AuNP to expand the scope of their utility.  We designed and synthesized AuNP probes displaying an azide group, a photoreactive group, a hydrophilic spacer group.  Any small-molecule ligand of interest with an alkyne tag can be immobilized on these clickable probes in one step by click chemistry.  Using a bovine carbonic anhydrase (BCAⅡ) inhibitor with an alkyne tag, we demonstrated that both copper-promoted azide-alkyne cyclization and photoaffinity labeling can be performed at nanomolar concentrations of AuNP probes.  The effectiveness of our new clickable photoaffinity probes were evaluated by SDS-PAGE following the thiol-exchange reaction and by direct application of MALDI-MS.