Multistructure sequencing of N-linked fetuin glycans by capillary gel electrophoresis and enzyme matrix digestion
Multistructure sequencing of N-linked fetuin glycans by capillary gel electrophoresis and enzyme matrix digestion
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DOI：
10.1002/elps.1150180719
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发表时间：
1997-06-01
期刊：
影响因子：
2.9
通讯作者：
Guttman, A
中科院分区：
文献类型：
--
作者：
Guttman, A
描述了通过使用精心设计的外切糖苷酶基质对N-连接胎球蛋白聚糖进行特异性酶促消化，并结合高效毛细管凝胶电泳分离合并的寡聚物进行寡糖测序。由于毛细管凝胶电泳具有极高的分离能力和出色的重现性，在大多数情况下，不需要分离单个寡糖，从而能够从释放的聚糖池进行多结构测序。通过比较分离的外切糖苷酶消化片段与已知大小的麦芽寡糖的位置，容易计算相对迁移时间和迁移位移。因此，可以高置信度地提出释放的聚糖库中每种低聚糖的特定序列。此外，通过使用高灵敏度的激光诱导荧光检测，可以从皮摩尔量的纯化糖蛋白获得完整的序列信息。
Oligosaccharide sequencing by specific enzymatic digestion of N-linked fetuin glycans using a carefully designed exoglycosidase matrix in conjunction with separation of the combined digests by high performance capillary gel electrophoresis is described. Due to the extremely high separation power and excellent reproducibility of capillary gel electrophoresis, in most instances no isolation of the individual oligosaccharides is necessary, enabling multistructure sequencing from a released glycan pool. By comparing the positions of the separated exoglycosidase digest fragments to maltooligosaccharides of known size, relative migration times and migration shifts are easily calculated. Hence, the particular sequence of each oligosaccharide in a released glycan pool can be proposed with high confidence. Additionally, with the use of high sensitivity laser-induced fluorescence detection, complete sequence information can be attained from picomolar amounts of purified glycoproteins.