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A proteomics approach to the cell‐surface interactome using the enzyme‐mediated activation of radical sources reaction

We previously reported a simple method to analyze the interaction of cell‐surface molecules in living cells. This method termed enzyme‐mediated activation of radical sources (EMARS) is featured by radical formation of the labeling reagent by horseradish peroxidase (HRP). Herein, we propose an approach to the cell‐surface molecular interactome by using combination of this EMARS reaction and MS‐based proteomics techniques. In the current study, we employed a novel labeling reagent, fluorescein‐conjugated arylazide. The fluorescein‐tagged proteins resulting from the EMARS reaction were directly detected in the electrophoresis gels with a fluorescence image analyzer. These products were also purified and concentrated by immunoaffinity chromatography with anti‐fluorescein antibody‐immobilized resins. The purified fluorescein‐tagged proteins were subsequently subjected to an MS‐based proteomics analysis. Analysis using HRP‐conjugated cholera toxin subunit B, which recognizes a lipid raft marker, ganglioside GM1, revealed 30 membrane and secreted proteins that were candidates for the cell‐surface molecules coclustering with GM1. The proposed approach will provide a clue to study functional molecular interactions in a variety of biological events on the cell surface.