SEPARATION OF POSITIONAL ISOMERS OF OLIGOSACCHARIDES AND GLYCOPEPTIDES BY HIGH-PERFORMANCE ANION-EXCHANGE CHROMATOGRAPHY WITH PULSED AMPEROMETRIC DETECTION
SEPARATION OF POSITIONAL ISOMERS OF OLIGOSACCHARIDES AND GLYCOPEPTIDES BY HIGH-PERFORMANCE ANION-EXCHANGE CHROMATOGRAPHY WITH PULSED AMPEROMETRIC DETECTION
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DOI：
10.1073/pnas.85.10.3289
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发表时间：
1988-05-01
影响因子：
11.1
通讯作者：
TOWNSEND, RR
中科院分区：
文献类型：
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作者：
HARDY, MR;TOWNSEND, RR
在碱性条件下（pH = 1.5）进行高效阴离子交换（HPAE）色谱分析。13)已经发现根据分子大小、糖组成和单糖单元的键合有效地分离中性寡糖（三糖至十一糖）。该方法能够解析中性寡糖的1 → 3、1 → 4和1 → 6位置异构体，其被定义为具有相同数目、类型、序列和单糖的端基异构体构型，但单糖的键合位置不同。从不同寡糖的相关结构特征和保留时间，我们推断，至少有两个因素是可操作的，以确定通过这种类型的色谱的寡糖的上级分辨率：（i）羟基的相对酸度和（ii）的寡糖的含氧阴离子的固定相的官能团的可及性。未观察到归因于变旋的峰分裂。还原低聚糖比它们的还原对应物保留得多。Fuc（α 1 -3）与利戈替尼的GlcNAc的连接显著降低了保留时间。两种支化单糖的位置异构体（其相差1 → 6，1 → 4键）被广泛分离。四糖和含十一糖的糖肽的1 → 3和1 → 4位置异构体的分离证明，与以前的通过反相或胺键合固定相的色谱方法相比，HPAE的分离度有显著的提高。皮摩尔量的未衍生化的寡糖已被检测三脉冲安培检测，产生了类似的反应，为广泛的结构。通过使用检测器响应或1H NMR定量牛胎球蛋白中的两种三触角糖肽具有可比性。N-聚糖酶催化的两个1-fdarw. 4和1 →牛胎球蛋白的胰蛋白酶糖肽的十一糖的3个位置异构体可以通过直接注射酶混合物到色谱中来观察和定量。
High-performance anion-exchange (HPAE) chromatography under alkaline conditions (pH .simeq. 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.fwdarw.3, 1.fwdarw.4, and 1.fwdarw.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 ligosaccharides markedly decreased retention times. Positional isomers of two branched monosaccharides, which differed by 1.fwdarw.6, 1.fwdarw.4 linkages, were widely separated. The separation 1.fwdarw.3, and 1.fwdarw.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 .fwdarw. 4 and 1 .fwdarw. 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 chromatography.