High-Sensitivity Analysis of Naturally Occurring Sugar Chains, Using a Novel Fluorescent Linker Molecule

Abstract

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, welldesigned 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 wellestablished 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 kg 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 goldcoated 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. The carbohydrates that make up proteoglycans, glycoproteins or glycolipids are responsible for many biological functions and play crucial roles in cellular binding and signalling (1). However, because of their structural complexity, the methods for studying sugar chains are more challenging than that for DNA, RNA or proteins. The numerous isomeric and anomeric configurations of sugar chains, as well as the difficulties in isolating sufficient quantities of naturally occurring sugars, make binding analysis and structure-function studies challenging.For the structural analysis of naturally occurring sugar chains, fluorescent labelling of the sugars has been one popular technique (2). Recently, mass spectrometry (MS) has been used for structural analysis of sugar structures, thanks to the development of structurally well-defined standards (3, 4). Surface plasmon resonance (SPR) methodology is also a very effective method to quantify binding interactions between sugar-chains and lectins or viruses in real time, because it is a non-destructive technology that does not require large quantities of the often scarce materials to be studied (5-9). We have previously reported the development of the 'sugar chip', in which defined sugar chains are immobilized on an SPR sensor chip using our specialized linker molecules (10,11). But the puri...

Visual Detection of Human Antibodies Using Sugar Chain-Immobilized Fluorescent Nanoparticles: Application as a Point of Care Diagnostic Tool for Guillain-Barré Syndrome

“…Sugar chain-ligand conjugates 1 and 2 were used as the GM1 sugar moieties. The conjugates were prepared by reductive amination of a chemically synthesized GM1-Glc sugar component [ 22 ] and a commercially available GM1 sugar component (ELICITYL, Crolles, France), respectively, with a fluorescent linker molecule (f-mono, Fig 1 ) [ 21 ]. The preparation of SFNPs with these conjugates was performed using our previous method, with a slight modification ( Fig 1B ).…”

“…GM1-Glc-f-mono was synthesized according to the methods described in our previous report [ 21 ]. The GM1-Glc sugar chain [ 22 ] (1.0 mg, 0.86 μmol) and f-mono [ 21 ] (0.28 mg, 1.1 μmol) were dissolved in water (20 μL) and N , N -dimethylacetamide (30 μL), respectively. Then, the GM1-Glc solution and acetic acid (6 μL) were added to the f-mono solution.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2009–2010

“…Key to this method is the use of linker compounds for immobilization of the sugar chains to the gold‐coated SPR chip. Sato et al () have developed a novel linker molecule, named “f‐mono,” which combines a linker with a fluorescent moiety to allow high sensitivity monitoring of the glycans. Since the molecule ( 32 ) contains a 2,5‐diaminopyridyl group and a thioctic acid group, conjugation with sugar chains can be achieved by the reductive amination reaction.…”

Derivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry

“…The derivative has the useful property of being partially reduced during mass spectral analysis (ESI), the normal [M+H] + ions are accompanied by a second ion ([M+H+2H] + ) appearing two mass units higher. The doublet peaks enable derivatized glycans in a mixture to be easily distinguished from other compounds [215]. Lipids such as 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE, 54) have also been coupled via reductive amination and, in order to avoid using a carbohydrate with an open reducing terminal ring, the related compound (55) has been coupled via oxime formation [216,217].…”