Ultra-sensitive Analysis of Carbohydrates |
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(First version published:Dec.15, 1999) | |||||||||||||||||||||||||||||
Characterization of glycans in glycoproteins and glycosaminoglycans involves enzymatic or chemical release, derivatization with fluorescent tag, and isolation followed by structural determination. Separation or analysis is the key step in these processes, and has often been attained by using high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE). Highly specific and sensitive detection is required for the analysis of micro amounts of oligosaccharides derived from the glycoconjugates. Detection based on fluorescence offers the best choice for the detection of carbohydrates at high sensitivity. The most characteristic feature of glycans is the possession of reactive aldehyde or keto groups in their reducing ends. These carbonyl groups are often labeled with fluorescent amines by reductive amination (Fig. 1). Numerous numbers of reagents have been reported for this purpose (Fig. 2). Labeling using 2-aminopyridine (AP), originally reported by Hase et al.(1) has been developed to multidimensional mapping by Takahashi et al.(2,3). The structures of glycoprotein glycans were easily confirmed by plotting the elution indexes obtained by separation of pmol amounts of the PA-labeled glycans on reversed phase, amide-bonding phase and ion exchange DEAE columns. Diffusion of sample components in the LC system hampers sensitive detection. Capillary electrophoresis (CE) using a very narrow capillary (~50 µm i.d.) has come to the front. Separated analytes are often fluorimetrically detected by irradiating a laser beam. Derivatization with 8-aminopyrene-1,3,6-trisulfonate (APTS) is most attractive in CE analysis, because the sulfonate groups force the glycan derivatives to anode with high velocity and thus decreases the analysis time (to often less than 15 min). The excitation wavelength of APTS is shifted from 424 nm to 456 nm by conversion to APTS-glycans, which enables sensitive detection by an argon laser-induced fluorimetric CE system without the removal of excess APTS reagent from the reaction mixture. Guttman et al. reported fine resolution of glycans derived from various glycoproteins(4). Their detection limits reached the 10-18 mol level. The APTS derivatization was applied to the simultaneous automated analysis of glycoprotein glycans using a DNA sequencer. Glycans were released from glycoproteins and derivatized with APTS on a 96-well plate automatically and analyzed by a multi-capillary CE system(5). This method seems to be suitable for the routine analysis or quality control of glycoprotein glycans. |
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Reductive amination reaction is not suitable for quantitative labeling of ketoses and sialic acids. Benzene-diamine derivatives react with α-keto groups of sialic acids to give strong fluorescent quinoxaline compounds. Hara et al. applied 1,2-diamino-4,5-methylenedioxabenzene (DMB) to the reversed phase HPLC analysis of sialic acids(7). This reaction requires an excess amount of reducing agent to prevent oxidation of DMB derivatives. Anumula et al. reported the derivatization with o-phenylenediamine as a more convenient and sensitive method(7). The analysis of glycan samples containing large amounts of proteins or peptides often meets difficulty in specific detection even though glycans are labeled with sensitive reagents. Shimaoka et al. proposed the usage of a saccharide trapping column(8). Glycans are covalently bonded to amino- or hydrazine groups of polymer and trapped, whereas other materials are removed simply by elution with appropriate solvents. Trapped carbohydrates are released as fluorimetric hydrazones which enables sensitive detection by fluorimetric detection HPLC or LC-MS for structural analysis. The method will become a useful strategy for the analysis of minute amounts of glycans in biological samples. |
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Masahiro Yodoshi and Shigeo Suzuki (Faculty of Pharmaceutical Sciences, Kinki University) |
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Apr. 23, 2007 | |||||||||||||||||||||||||||||
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