Target Protein Identification on Photocatalyst-Functionalized Magnetic Affinity Beads

Although various affinity chromatography and photoaffinity labeling methods have been developed for target protein identification of bioactive molecules, it is often difficult to detect proteins that bind the ligand with weak transient affinity using these techniques. We have developed single electron transfer–mediated tyrosine labeling using ruthenium photocatalysts. Proximity labeling using 1-methyl-4-aryl-urazole (MAUra) labels proteins in close proximity to the photocatalyst with high efficiency and selectivity. Performing this labeling reaction on affinity beads makes it possible to label proteins that bind the ligand with weak transient affinity. In this article, novel protocols are described for target protein identification using photocatalyst proximity labeling on ruthenium photocatalyst-functionalized magnetic affinity beads. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis of ruthenium photocatalyst

Basic Protocol 2: Synthesis of azide- or desthiobiotin-conjugated labeling reagents

Basic Protocol 3: Preparation of photocatalyst and ligand-functionalized affinity beads

Basic Protocol 4: Target protein labeling in cell lysate

Basic Protocol 5: Enrichment of labeled proteins with MAUra-DTB for LC-MS/MS analysis

Basic Protocol 6: 2D-DIGE analysis of fluorescence-labeled proteins