Analysis of glycoprotein oligosaccharides using high-pH anion exchange chromatography.
Analysis of glycoprotein oligosaccharides using high-pH anion exchange chromatography.
复制标题
使用高 pH 阴离子交换色谱法分析糖蛋白寡糖。
DOI：
10.1093/glycob/1.2.139
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发表时间：
1991
期刊：
影响因子：
4.3
通讯作者：
Hardy,MR
中科院分区：
文献类型：
--
作者：
Townsend,RR;Hardy,MR
蛋白质糖基化是翻译过程中和翻译后发生的主要修饰。碳水化合物被发现与蛋白质的AsnXxxSer（Thr）N-连接（Snider，1984; Kornfeld和Kornfeld，1985），很少出现在AsnXxxCys序列中（Titani等，1986），O-连接至Ser或Thr残基（萨德勒，1984年;哈特等人，1989年），并通过糖基磷脂酰肌醇（GPI）锚中的乙醇胺磷酰基连接到羧基末端（Ferguson和威廉姆斯，1988年）。这些“翻译后”修饰在图1中图示。两个主要类别的N-连接结构，高甘露糖（寡甘露糖苷）和复杂的（乳糖胺）型，GalNAc和GlcNAc型O-糖基化，和GPI锚的核心结构的代表。糖基化的一个标志，即使在单个肽位点，是结构异质性。因此，从糖蛋白中分离的寡糖是混合物，通常是密切相关的异构体化合物。单个寡糖之间的结构差异可能涉及大小、组成、序列、端基异构性、连接位置（1-· 2、1-> 3、1-· 4或1-> 6）和分支构型。根据上述特征拆分寡糖链的分离方法是结构解析的重要组成部分，是理解蛋白质糖基化生物学的基础。尽管有许多可用于碳水化合物的色谱技术（本田，1984;希克斯，1988），但很少有人证明可用于从糖蛋白中干净地分离许多中性和阴离子（如唾液酸化、磷酸化和硫酸化）异构体形式。胺键合的HPLC已经显示出用于分离中性的一些异构体的实用性（Blanken等人，1985）和唾液酸化低聚糖（绿色和Baenziger，1986）。中性吡啶基胺化低聚糖，其包括键和支链异构体形式，已经使用反相和正相高效液相色谱的组合进行分离（Hase等人，1987; Tomiya等人，1988年）。连续凝集素亲和层析（Merkle和Cummings，1987; Osawa和Tsuji，1987;绿色和Baenziger，1989）和高效配体亲和层析（Zopf等人，1989）是根据结构特征分离寡糖的有力方法，所述结构特征通常涉及键和分支构型。最近，高pH阴离子交换色谱法（HPAEC）已经显示出能够解析中性和阴离子寡糖两者的键和分支异构体（Chen等人，1988;哈代和汤森，1988;汤森等人，1989 a; Wang和Zopf，1989）。现在有多个报道详细描述了在微膜季铵树脂上分离糖蛋白和相关寡糖作为它们的含氧阴离子的效用（汤森等人，1988，1989 b，1991;哈代和汤森，1989; Anumula和Taylor，1991; Basa和Spellman，1990;埃尔南德斯等人，1990; Pfeiffer等人，1990; Wang等人，1990; Yet和Wold，1990）。HPAEC与脉冲安培检测（PAD）（约翰逊和LaCourse，1990）和用于糖蛋白分析的放射性检测的组合为理解蛋白质的糖生物学提供了新的工具。
Protein glycosylation is a major modification which occurs during and after translation. Carbohydrate has been found N-linked to proteins at AsnXxxSer (Thr)(Snider, 1984; Kornfeld and Kornfeld, 1985) and rarely at AsnXxxCys sequons (Titani et al, 1986), O-linked to Ser or Thr residues (Sadler, 1984; Hart et al, 1989), and attached to the carboxyl terminal via an ethanolamine phosphoryl group in glycosylphosphatidylinositol (GPI) anchors (Ferguson and Williams, 1988). These'post-translational'modifications are schematized in Figure 1. Two major classes of N-linked structures, high-mannose (oligomannosidic) and complex (lactosamine) type, GalNAc and GlcNAc-type O-glycosylation, and the core structure of GPI anchors are represented. A hallmark of glycosylation, even at individual peptide loci, is structural heterogeneity. Thus, oligosaccharides which are isolated from glycoproteins are mixtures, often of closely related isomeric compounds. Structural differences among individual oligosaccharides may involve size, composition, sequence, anomerity, linkage position (1-• 2, 1-> 3, 1-• 4, or 1-> 6) and branching configurations. Separation methods which resolve oligosaccharide chains according to the above features are an essential part of structural elucidation—the underpinning for understanding the biology of protein glycosylation. Despite the many chromatographic techniques that are available for carbohydrates (Honda, 1984; Hicks, 1988), few have demonstrated utility for cleanly separating many of the neutral and anionic (eg sialylated, phosphorylated and sulphated) isomeric forms from glycoproteins. Aminebonded HPLC has shown utility for separating some isomers of neutral (Blanken et al., 1985) and sialylated oligosaccharides (Green and Baenziger, 1986). Neutral pyridylaminated oligosaccharides, which included linkage and branched isomeric forms, have been separated using a combination of reversed and normal phase high-performance liquid chromatography (Hase et al., 1987; Tomiya et al., 1988). Serial lectin affinity chromatography (Merkle and Cummings, 1987; Osawa and Tsuji, 1987; Green and Baenziger, 1989) and high-performance ligand affinity chromatography (Zopf et al., 1989) are powerful methods for separating oligosaccharides according to structural features which often involve linkages and branching configurations. Most recently, high-pH anion-exchange chromatography (HPAEC) has been shown to resolve linkage and branch isomers of both neutral and anionic oligosaccharides (Chen et al., 1988; Hardy and Townsend, 1988; Townsend et al., 1989a; Wang and Zopf, 1989). There are now multiple reports which detail the utility of separating glycoprotein and related oligosaccharides as their oxyanions on micropellicular quaternary ammonium resins (Townsend et al., 1988, 1989b, 1991; Hardy and Townsend, 1989; Anumula and Taylor, 1991; Basa and Spellman, 1990; Hernandez et al, 1990; Pfeiffer et al, 1990; Wang et al, 1990; Yet and Wold, 1990). The combination of HPAEC with pulsed amperometric detection (PAD)(Johnson and LaCourse, 1990) and radiometric detection for the analysis of glycoproteins has provided a new tool toward understanding the glycobiology of proteins.