title: A proteomics approach to the cell-surface interactome using the enzyme-mediated activation of radical sources reaction.
abstract: 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.
authors: Department of Biochemistry, Kochi University Medical School, Nankoku, Kochi, Japan.
authors: Songlin
authors: S
authors: Jiang
authors: Norihiro
authors: N
authors: Kotani
authors: Tomoko
authors: T
authors: Ohnishi
authors: Arisa
authors: A
authors: Miyagawa-Yamguchi
authors: Masayuki
authors: M
authors: Tsuda
authors: Ryusuke
authors: R
authors: Yamashita
authors: Yoshihito
authors: Y
authors: Ishiura
authors: Koichi
authors: K
authors: Honke
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005677:G(M1) Ganglioside / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D006825:Hybridomas; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics; D013194:Staining and Labeling
chemical_list: D001386:Azides; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D020543:Proteome; D005677:G(M1) Ganglioside; D006735:Horseradish Peroxidase; D019793:Fluorescein