Journal of Biochemistry Advance Access originally published online on March 6, 2009
Journal of Biochemistry 2009 146(1):33-41; 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; 3Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, Japan; 4Department of Surgery, VA Puget Sound HCS and The University of Washington School of Medicine, Seattle, WA, USA; and 5SUDx-Biotec Corp., 1-42-1, Shiroyama, Kagoshima, 890-0013, Japan
*To whom correspondence should be addressed. Tel: +81-99-285-8369, 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 analyse 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, non-destructive 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 labelling reagents are essential when analysing 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 µg of FLC can be easily purified using HPLC with a fluorescent detector. MS and MS/MS analysis of the FLC is also possible. As 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.
Key Words: immobilization, sugar chain, high sensitivity, analysis, fluorescence, linker molecule, mass spectrometry
Abbreviations:
DMAc, N, N-dimethyl acetoamide; aoWS, N
-((aminooxy)acetyl)tryptophanylarginine methyl ester