Honda S, Makino A, Suzuki S, Kakehi K
Faculty of Pharmaceutical Sciences, Kinki University, Higashi-osaka, Japan.
Anal Biochem. 1990 Dec;191(2):228-34. doi: 10.1016/0003-2697(90)90212-r.
The oligosaccharides in ovalbumin as a glycoprotein model were released with anhydrous hydrazine, and reductively pyridylaminated after re-N-acetylation. The derivatives were analyzed by capillary zone electrophoresis (CZE) with on-column fluorometric detection. Direct CZE could separate the derivatives on the basis of the degree of polymerization, giving five peaks of hepta-, octa-, nona-, deca-, and undecasaccharides. Coelectrophoresis with the standard mixture of isomaltooligosaccharide derivatives was effective for peak assignment. CZE as borate complexes allowed separation on the basis of structural difference, especially in the peripheral monosaccharide residues. Peaks were tentatively assigned to the derivatives of reported oligosaccharides by comparing their relative mobilities with those of the chromatographic fractions obtained by using the ODS and Dowex 50W x 2 columns. These two modes gave excellent separation and were complementary to each other. Although the actual amount analyzed in the capillary tube was quite small (ca. 5 ng as carbohydrates), a larger amount (ca. 25 micrograms as carbohydrates) was required to make sample concentration sufficiently high to be detected by a modification of a commercial fluoromonitor for HPLC.
以卵清蛋白作为糖蛋白模型，其寡糖用无水肼释放，并在重新进行N - 乙酰化后进行还原吡啶氨基化。衍生物通过带柱上荧光检测的毛细管区带电泳（CZE）进行分析。直接CZE可根据聚合度分离衍生物，得到七糖、八糖、九糖、十糖和十一糖的五个峰。与异麦芽寡糖衍生物标准混合物进行共电泳对峰的归属有效。作为硼酸盐络合物的CZE能够基于结构差异进行分离，特别是在外围单糖残基方面。通过比较它们与使用ODS和Dowex 50W x 2柱获得的色谱馏分的相对迁移率，将峰初步归属于报道的寡糖的衍生物。这两种模式分离效果极佳且相互补充。尽管毛细管中实际分析的量非常小（约5 ng碳水化合物），但需要更大的量（约25 μg碳水化合物）以使样品浓度足够高，以便通过对商用HPLC荧光监测仪进行改装来检测。
