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首页> 外文期刊>Toxicon: An International Journal Devoted to the Exchange of Knowledge on the Poisons Derived from Animals, Plants and Microorganisms >Structural characterization of N-linked oligosaccharides of Defibrase from Agikistrodon acutus by sequential exoglycosidase digestion and MALDI-TOF mass spectrometry
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Structural characterization of N-linked oligosaccharides of Defibrase from Agikistrodon acutus by sequential exoglycosidase digestion and MALDI-TOF mass spectrometry

机译：连续糖外切酶消化和MALDI-TOF质谱分析a蛇脱纤维酶N-连接寡糖的结构特征

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摘要

Detailed structures of N-linked oligosaccharides of Defibrase, a highly active thrombin like enzyme (TLE) purified from the venom of Agkistrodon acutus, were successfully characterized using MALDI-TOF mass spectrometry in combination with sequential exoglycosidase digestion. Monosaccharide composition analysis was performed by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Galactose(Gal), mannose(Man), fucose(Fuc), N-acetylglucosamine (GlcNAc), and sialic acid (Neu5Ac) was detected and the total carbohydrate content was about 19.4% (w/w). The N-linked oligosaccharides were released by treatment with PNGase F, fluorescent labeled with 2-aminobenzamide, and fractionated by high performance liquid chromatography (HPLC). The main oligosaccharide fractions were collected and further digested with an array of exoglycosidase mixtures, and subsequent MALDI TOF MS analysis of the resulting products yielded information about structural features of the oligosaccharide. The combined data revealed the presence of five distinct oligosaccharide structures in Defibrase, which are mainly complex or hybrid type, with a small amount of oligomannosidic type. The complex type oligosaccharides are mostly tri-or bi-antennary and the hybrid oligosaccharides are all bi-antennary. Most oligosaccharides are also found to be fucosylated.
机译：使用MALDI-TOF质谱结合序贯外切糖苷酶消化法成功地表征了脱纤维酶的N-连接寡糖的详细结构。通过高效阴离子交换色谱和脉冲安培检测（HPAEC-PAD）进行单糖成分分析。检测到半乳糖（Gal），甘露糖（Man），岩藻糖（Fuc），N-乙酰氨基葡萄糖（GlcNAc）和唾液酸（Neu5Ac），总碳水化合物含量约为19.4％（w / w）。通过用PNGase F处理释放N-连接的寡糖，用2-氨基苯甲酰胺荧光标记，并通过高效液相色谱（HPLC）分离。收集主要的寡糖馏分，并用一系列糖苷外切酶混合物进一步消化，然后对所得产物进行MALDI TOF MS分析，得出有关寡糖结构特征的信息。合并的数据显示，在去纤酶中存在五个不同的寡糖结构，主要是复合型或杂合型，以及少量的寡甘露糖苷型。复杂类型的低聚糖主要是三触角或双触角，而杂合的低聚糖都是双触角。还发现大多数寡糖是岩藻糖基化的。

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