These authors contributed equally to this work.
A proteomics approach to the cell-surface interactome using the enzyme-mediated activation of radical sources reaction†
Article first published online: 9 DEC 2011
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Volume 12, Issue 1, pages 54–62, January 2012
How to Cite
Jiang, S., Kotani, N., Ohnishi, T., Miyagawa-Yamguchi, A., Tsuda, M., Yamashita, R., Ishiura, Y. and Honke, K. (2012), A proteomics approach to the cell-surface interactome using the enzyme-mediated activation of radical sources reaction. Proteomics, 12: 54–62. doi: 10.1002/pmic.201100551
Colour online: See the article online to view Fig. 2 in colour.
- Issue published online: 5 JAN 2012
- Article first published online: 9 DEC 2011
- Accepted manuscript online: 22 NOV 2011 06:11AM EST
- Manuscript Accepted: 27 OCT 2011
- Manuscript Revised: 25 OCT 2011
- Manuscript Received: 18 OCT 2011
- Institute of Development, Aging and Cancer, Tohoku University for generous gift. Grant Number: TS2/16
- JSPS KAKENHI. Grant Number: 22659060
- Japan Science and Technology Agency and the Naito Foundation
- Cell biology;
- Cell surface;
- Lipid raft;
- Molecular interaction
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.