Guttman A
Beckman Instruments, Inc., Fullerton, CA, USA.
Electrophoresis. 1997 Jun;18(7):1136-41. doi: 10.1002/elps.1150180719.
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.
本文描述了通过使用精心设计的外切糖苷酶矩阵对N-连接胎球蛋白聚糖进行特异性酶切，并结合高效毛细管凝胶电泳对混合消化产物进行分离来进行寡糖测序的方法。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，在大多数情况下无需分离单个寡糖，从而能够从释放的聚糖池中进行多结构测序。通过将分离的外切糖苷酶消化片段的位置与已知大小的麦芽寡糖进行比较，可以轻松计算相对迁移时间和迁移位移。因此，可以高度自信地推测释放的聚糖池中每种寡糖的特定序列。此外，通过使用高灵敏度激光诱导荧光检测，从皮摩尔量的纯化糖蛋白中即可获得完整的序列信息。
