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Molecular & Cellular Proteomics 8:2256-2265, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Identification of Heparin-binding Sites in Proteins by Selective Labeling^*,

Alessandro Ori, Paul Free, José Courty, Mark C. Wilkinson^,¶ and David G. Fernig^,¶,||

From the School of Biological Sciences and Centre for Glycobiology, University of Liverpool, Liverpool L69 7ZB, United Kingdom and
Laboratoire de Recherche sur la Croissance Cellulaire, la Réparation et la Régénération Tissulaires (CRRET), CNRS UMR 7149, Université Paris XII, 94010 Créteil Cedex, France

Heparan sulfate proteoglycans are key regulators of complex molecular networks due to the interaction of their sugar chains with a large number of partner proteins, which in humans number more than 200 (Ori, A., Wilkinson, M. C., and Fernig, D. G. (2008) The heparanome and regulation of cell function: structures, functions and challenges. Front. Biosci. 13, 4309–4338). We developed a method to selectively label residues involved in heparin binding that matches the requirements for medium/high throughput applications called the "Protect and Label" strategy. This is based on the protection against chemical modification given by heparin/heparan sulfate to the residues located in the heparin-binding site. Thus, analysis of fibroblast growth factor-2 bound to heparin and incubated with N-hydroxysuccinimide acetate showed that lysines involved in the sugar binding are protected against chemical modification. Moreover following release from heparin, the protected lysine side chains may be specifically labeled with N-hydroxysuccinimide biotin. After protein digestion, the biotinylated peptides were readily isolated and identified by MALDI-Q-TOF mass spectrometry. The analysis of labeled peptides obtained from three well characterized heparin-binding proteins with very different heparin-binding sites, fibroblast growth factor-2, platelet factor-4, and pleiotrophin demonstrates the success of this new approach, which thus provides a rapid and reliable procedure to identify heparin-binding sites.

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