Hardy M R, Townsend R R
Department of Biology, Johns Hopkins University, Baltimore, MD 21218.
Proc Natl Acad Sci U S A. 1988 May;85(10):3289-93. doi: 10.1073/pnas.85.10.3289.
High-performance anion-exchange (HPAE) chromatography under alkaline conditions (pH congruent to 13) has been found to efficiently separate neutral oligosaccharides (triose to undecaose) according to molecular size, sugar composition, and linkage of monosaccharide units. The method was able to resolve 1----3, 1----4, and 1----6 positional isomers of neutral oligosaccharides, which are defined as having the same number, type, sequence, and anomeric configurations of monosaccharides but differing in the linkage position of a single sugar. From correlating structural features of different oligosaccharides and retention times, we deduced that at least two factors are operative to determine the superior resolution of oligosaccharides by this type of chromatography: (i) the relative acidities of the hydroxyl groups and (ii) the accessibility of oxyanions of the oligosaccharides to the functional groups of the stationary phase. Splitting of peaks attributable to mutarotation was not observed. Reducing oligosaccharides were much more retained than their reduced counterparts. Linkage of Fuc(alpha 1-3) to GlcNAc of oligosaccharides markedly decreased retention times. Positional isomers of two branched monosaccharides, which differed by 1----6 and 1----4 linkages, were widely separated. The separation of 1----3 and 1----4 positional isomers of both tetrasaccharides and glycopeptides containing undecasaccharides demonstrated the significant improvement in resolution of HPAE compared to previous chromatographic methods by either reverse-phase or amine-bonded stationary phases. Picomole quantities of underivatized oligosaccharides have been detected by triple-pulse amperometric detection, which produced similar responses for a wide range of structures. Quantification of two triantennary glycopeptides from bovine fetuin by using either detector response or 1H NMR was comparable. The N-glycanase-catalyzed release of two 1----4 and 1----3 positional isomers of an undecasaccharide from a tryptic glycopeptide of bovine fetuin could be observed and quantified by direct injection of the enzyme mixture into the chromatograph.
已发现，在碱性条件（pH值等于13）下的高效阴离子交换（HPAE）色谱法能够根据分子大小、糖组成和单糖单元的连接方式有效地分离中性寡糖（丙糖至十一糖）。该方法能够分离中性寡糖的1→3、1→4和1→6位置异构体，这些异构体被定义为具有相同数量、类型、序列和单糖的异头构型，但在单个糖的连接位置上有所不同。通过关联不同寡糖的结构特征和保留时间，我们推断至少有两个因素决定了此类色谱法对寡糖的卓越分离效果：（i）羟基的相对酸度；（ii）寡糖的氧阴离子与固定相官能团的可及性。未观察到因变旋导致的峰分裂。还原性寡糖比其还原后的对应物保留时间长得多。寡糖中Fuc(α1-3)与GlcNAc的连接显著缩短了保留时间。两种分支单糖的位置异构体，其连接方式分别为1→6和1→4，被广泛分离。四糖和含十一糖的糖肽的1→3和1→4位置异构体的分离表明，与先前使用反相或胺键合固定相的色谱方法相比，HPAE的分离效果有了显著提高。通过三脉冲安培检测法已检测到皮摩尔量的未衍生化寡糖，该方法对广泛的结构产生相似的响应。使用检测器响应或1H NMR对来自牛胎球蛋白的两种三触角糖肽进行定量的结果相当。通过将酶混合物直接注入色谱仪中，可以观察到并定量N-糖苷酶催化从牛胎球蛋白的胰蛋白酶糖肽中释放出的十一糖的两种1→4和1→3位置异构体。
