Journal of Biochemistry Advance Access published online on March 6, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp041
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High sensitivity analysis of naturally occurring sugar-chains, using a novel fluorescent linker molecule
1Department of Nanostructure and Advanced Materials, 2Department of Bioengineering, Graduate School of Science and Engineering, Kagoshima University, 1-21-40, Kohrimoto, Kagoshima 890-0065, Japan, 3Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 4Department of Surgery, VA Puget Sound HCS and The University of Washington School of Medicine,, Seattle WA, USA., 5SUDx-Biotec Corp., 1-42-1, Shiroyama, Kagoshima, 890-0013, Japan.
* Address correspondence to: Yasuo Suda, Department of Nanostructure and Advanced Materials, Graduate School of Science and Engineering, Kagoshima University, 1-21-40, Kohrimoto, Kagoshima 890-0065, Japan; Tel & Fax: +81-99-285-8369; E-mail: ysuda{at}eng.kagoshima-u.ac.jp
Received February 16, 2009; Accepted February 26, 2009
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Abstract |
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To analyze the binding of sugar-chains to proteins, viruses, and cells, the surface plasmon resonance (SPR) technique is very convenient and effective because it is a real time, nondestructive detection system. Key to this method is linker compounds for immobilization of the sugar-chains to the gold coated chip for SPR. Also, well-designed fluorescent labeling reagents are essential when analyzing the structure of trace amounts of sugar-chains derived from natural sources, such as glycoproteins on the surface of specific cells. In this report, we developed a novel linker molecule, named "f-mono", which has both of these properties: simple immobilization chemistry and a fluorescent label. Since the molecule contains a 2,5-diaminopyridyl group and a thioctic acid group, conjugation with sugar-chains can be achieved using the well-established reductive amination reaction. This conjugate of sugar-chain and fluorescent linker (fluorescent ligand-conjugate, FLC) has fluorescent properties (ex. 335 nm, em. 380 nm), and as little as 1 g of FLC can be easily purified using HPLC with a fluorescent detector. MS and MS/MS analysis of the FLC is also possible. Because a +2 Da larger MS peak ([M+H+2]+ ion) was always associated with the theoretical MS peak ([M+H]+) (due to the reduction of the thioctic acid moiety), the MS peaks of the FLC were easily found, even using unfractionated crude samples. Immobilization of the FLC onto gold coated chips, and their subsequent SPR analyses were successively accomplished, as had been performed previously using non-fluorescent ligand conjugates.