MONOSACCHARIDE COMPOSITION ANALYSIS OF OLIGOSACCHARIDES AND GLYCOPROTEINS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY
MONOSACCHARIDE COMPOSITION ANALYSIS OF OLIGOSACCHARIDES AND GLYCOPROTEINS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY
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DOI：
10.1006/abio.1995.1294
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发表时间：
1995-05-20
影响因子：
2.9
通讯作者：
ONEILL, RA
中科院分区：
文献类型：
--
作者：
FU, DT;ONEILL, RA
介绍了一种简单、灵敏的高效液相色谱法（HPLC）分析低聚糖和糖蛋白中单糖组成的方法。在该方法中，首先使用优化的方法水解寡糖或糖蛋白以得到组成单糖，随后如先前由本田等人（Anal. 180，351-357，（1989））。使用专门为此目的开发的色谱柱，通过反相HPLC分离标记的单糖，通过245 nm处的紫外吸光度监测，并通过其相对于标准品的积分值进行定量。唾液酸是酸不稳定的酮基糖。因此，它们用神经氨酸酶或通过温和的酸水解释放，然后用神经氨酸醛缩酶转化为它们相应的甘露糖胺衍生物，然后如上所述对其进行PMP标记、分离和定量。包括N-乙酰基和N-羟乙酰基神经氨酸在内的单个唾液酸是良好的。该方法灵敏度高，检测灵敏度仅需1 pmol，采用一次酸水解和一组等摩尔单糖标准品，即可对寡糖和糖蛋白样品中的中性糖和氨基糖进行定量分析。类似地，唾液酸的定量同样适用于游离寡糖和糖蛋白样品。发现通过该方法测定的寡糖和糖蛋白的单糖组成高度准确，（C）1995 Academic Press，Inc.
A simple and sensitive high-performance liquid chromatography (HPLC)-based method for complete monosaccharide composition analysis of oligosaccharides and glycoproteins is described. In this method, an oligosaccharide or glycoprotein is first hydrolyzed using an optimized method to give the constituent monosaccharides, which are subsequently labeled with 1-phenyl-3-methyl-5-pyrazolone (PMP) as previously described by Honda ct al. (Anal. Biochem. 180, 351-357, (1989)). The labeled monosaccharides are separated by reverse-phase HPLC using a column developed especially for this purpose, monitored by uv absorbance at 245 nm, and quantitated by their integration values relative to standards, Sialic acids are acid-labile keto-sugars. They are, therefore, released with neuraminidases or by mild acid hydrolysis and then converted with neuraminic acid aldolase to their corresponding mannosamine derivatives, which are then PMP-labeled, separated, and quantitated as described above. Individual sialic acids including N-acetyl and N-glycolyl neuraminic acids are well. resolved and quantitated by this method, This method has proven to be highly sensitive, requiring only 1 pmol for reliable detection, Quantitative analysis of neutral and amino sugars from both oligosaccharide and glycoprotein samples can be achieved using one acid hydrolysis and a set of equal molar monosaccharide standards. Similarly, quantitation of sialic acids works equally well with both free oligosaccharide and glycoprotein samples, Monosaccharide compositions of oligosaccharides and glycoproteins determined by this method were found to be highly accurate, (C) 1995 Academic Press, Inc.