Guttman A, Ulfelder K W
Genetic BioSystems, Inc., San Diego, CA 92121, USA.
J Chromatogr A. 1997 Sep 26;781(1-2):547-54. doi: 10.1016/s0021-9673(97)00724-3.
This paper discusses oligosaccharide sequencing by consecutive enzymatic digestion of carbohydrates using an exoglycosidase array, followed by capillary electrophoresis separation of the digests. Because of the high resolving power and good reproducibility of capillary electrophoresis, multistructure sequencing of a complex glycan pool can be performed in most instances requiring no prior isolation of the individual oligosaccharides. High sensitivity laser-induced fluorescence detection enables acquisition of complete sequence information from several picomoles of glycoproteins. Comparison of the migration times of the exoglycosidase digest fragments to the maltooligosaccharide ladder, enables calculation of migration shifts, due to cleavage based on the actual exoglycosidases used. The particular sequence of each oligosaccharide in a glycan pool can be proposed with high confidence based on the migration time shifts of the various oligosaccharide structures. However, possible combinations of various sequence fragments may have very similar charge to hydrodynamic volume ratios, resulting in electrophoretic co-migration when a mixture of different oligosaccharides is sequenced together. Then, capillary electrophoresis separations of the resulting fragments should be evaluated after each digestion step. In the instances of complex separation profiles when multiple peaks are present, the evaluation of peak shifts can get very complicated and solved only with the aid of a software program. Data about the monosaccharide composition of the glycan pool provides useful information in designing the digestion enzyme matrix.
本文讨论了使用外切糖苷酶阵列对碳水化合物进行连续酶促消化，然后通过毛细管电泳分离消化产物来进行寡糖测序的方法。由于毛细管电泳具有高分辨率和良好的重现性，在大多数情况下，无需事先分离单个寡糖即可对复杂聚糖库进行多结构测序。高灵敏度激光诱导荧光检测能够从几皮摩尔糖蛋白中获取完整的序列信息。将外切糖苷酶消化片段与麦芽寡糖阶梯的迁移时间进行比较，可根据实际使用的外切糖苷酶的切割情况计算迁移偏移。基于各种寡糖结构的迁移时间偏移，可以高度自信地推断聚糖库中每个寡糖的特定序列。然而，各种序列片段的可能组合可能具有非常相似的电荷与流体动力学体积比，导致在对不同寡糖混合物进行测序时电泳共迁移。因此，在每个消化步骤后都应评估所得片段的毛细管电泳分离情况。在存在多个峰的复杂分离图谱的情况下，峰偏移的评估可能会非常复杂，并且只能借助软件程序来解决。关于聚糖库单糖组成的数据在设计消化酶矩阵时提供了有用的信息。
