Che F Y, Shao X X, Wang K Y, Xia Q C
Shanghai Institute of Biochemistry, Chinese Academy of Sciences.
Electrophoresis. 1999 Oct;20(14):2930-7. doi: 10.1002/(SICI)1522-2683(19991001)20:14<2930::AID-ELPS2930>3.0.CO;2-X.
A simple and highly sensitive capillary electrophoresis (CE) method for determining the content of N-acetylneuraminic acid (Neu5Ac) in glycoproteins was developed. Neu5Ac was derivatized with 2-aminoacridone (AMAC) by reductive amination, and the AMAC-Neu5Ac adduct could be readily separated from the other 11 AMAC-derivatized neutral and acidic monosaccharides usually present in glycoproteins by CE in a 0.3 mol/L borate buffer, pH 10.5, and detected at 260 nm. The derivatization of Neu5Ac was achieved at 55 degrees C for 4 h. AMAC-Neu5Ac was stable at 20 degrees C in the dark for at least 12 h while at room temperature it spontaneously converted into another substance with a lower electrophoretic mobility, which was identified as decarboxylated AMAC-Neu5Ac by high performance liquid chromatography - ion trap mass spectrometry (HPLC-ITMS). Concentration and mass of Neu5Ac as low as 1 micromol/L and 35 fmol could be detected. The linear correlation coefficient between the ratio of peak area to migration time of AMAC-Neu5Ac and the concentration of Neu5Ac ranging from 10 to 120 micromol/L was 0.9978 (n=8). This method was successfully applied to the analysis of sialic acid in human urinary trypsin inhibitor (hu-UTI), bovine alpha1-acid glycoprotein (alpha1-AGP) and recombinant human erythropoietin (rhu-EPO). By combination of CE and HPLC-ITMS we found that N-glycolylneuraminic acid (Neu5Gc) was present in bovine alpha1-AGP in addition to Neu5Ac, with a quantity comparable to that of the latter.
建立了一种简单且高灵敏度的毛细管电泳（CE）方法，用于测定糖蛋白中N-乙酰神经氨酸（Neu5Ac）的含量。Neu5Ac通过还原胺化反应与2-氨基吖啶酮（AMAC）衍生化，在pH 10.5的0.3 mol/L硼酸盐缓冲液中，通过CE可轻松将AMAC-Neu5Ac加合物与糖蛋白中通常存在的其他11种AMAC衍生化的中性和酸性单糖分离，并在260 nm处进行检测。Neu5Ac的衍生化在55℃下进行4小时。AMAC-Neu5Ac在20℃黑暗中至少12小时稳定，而在室温下它会自发转化为另一种电泳迁移率较低的物质，通过高效液相色谱-离子阱质谱（HPLC-ITMS）鉴定为脱羧AMAC-Neu5Ac。低至1 μmol/L和35 fmol的Neu5Ac浓度和质量均可被检测到。AMAC-Neu5Ac的峰面积与迁移时间之比与10至120 μmol/L的Neu5Ac浓度之间的线性相关系数为0.9978（n = 8）。该方法成功应用于人尿胰蛋白酶抑制剂（hu-UTI）、牛α1-酸性糖蛋白（α1-AGP）和重组人促红细胞生成素（rhu-EPO）中唾液酸的分析。通过CE和HPLC-ITMS相结合，我们发现除了Neu5Ac外，牛α1-AGP中还存在N-羟乙酰神经氨酸（Neu5Gc），其含量与后者相当。
