A novel tag-free probe for targeting molecules interacting with a flavonoid catabolite

•A novel tag-free probe for targeting molecules interacting with a flavonoid catabolite was developed.•Various labeled proteins were successfully detected by this probe with click chemistry.•A pulldown assay identified Keap1 and aryl hydrocarbon receptor as the target proteins of a flavonoid catabolite.

3,4-Dihydroxyphenylacetic acid (DOPAC) is one of the colonic microflora-produced catabolites of quercetin 4′-glucoside (Q4′G). Although the interaction of DOPAC with cellular proteins might be involved in its biological activity, the actual proteins have not yet been identified. In this study, we developed a novel tag-free DOPAC probe to label the targeted proteins by the copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) and verified its efficacy. Various labeled proteins were detected by the DOPAC probe with the azide labeled biotin and a horseradish peroxidase (HRP)-streptavidin complex. Furthermore, a pulldown assay identified Keap1 and aryl hydrocarbon receptor (AhR) as the target proteins for the phase 2 enzyme up-regulation.

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