PRECOLUMN LABELING OF REDUCING CARBOHYDRATES WITH 1-(PARA-METHOXY)PHENYL-3-METHYL-5-PYRAZOLONE - ANALYSIS OF NEUTRAL AND SIALIC ACID-CONTAINING OLIGOSACCHARIDES FOUND IN GLYCOPROTEINS
PRECOLUMN LABELING OF REDUCING CARBOHYDRATES WITH 1-(PARA-METHOXY)PHENYL-3-METHYL-5-PYRAZOLONE - ANALYSIS OF NEUTRAL AND SIALIC ACID-CONTAINING OLIGOSACCHARIDES FOUND IN GLYCOPROTEINS
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DOI：
10.1016/0003-2697(91)90099-f
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发表时间：
1991-12-01
影响因子：
2.9
通讯作者：
LEE, YC
中科院分区：
文献类型：
--
作者：
KAKEHI, K;SUZUKI, S;LEE, YC
建立了一种简便的柱前标记法，用1-（对甲氧基）苯基-3-甲基-5-吡唑啉酮（PMPMP）分析糖蛋白中的中性和含唾液酸寡糖。PMPMP与还原性寡糖在弱碱性条件下（pH 8.3）反应，形成2：1加合物（双-PMPMP衍生物）。寡糖中的唾液酸残基在反应过程中保持完整。胰蛋白酶糖肽用糖肽酶A消化释放寡糖，可直接用PMPMP衍生化，无需预先处理。标记的寡糖的分离进行了反相高效液相色谱法在C-18柱与乙腈水溶液，和位置异构体，如异构体三触角十四糖从牛胎球蛋白被完全解决。bis-PMPMP衍生物在碱性介质中不稳定，形成mono-PMPMP衍生物;然而，mono-PMPMP衍生物可以容易地再转化为原始的bis-PMPMP衍生物。该方法比还原吡啶胺化法简单，检测灵敏度可达亚纳摩尔。核糖核酸酶B（牛胰腺），卵清蛋白，甲状腺球蛋白（猪甲状腺），胎球蛋白（牛），和转铁蛋白（人）的寡聚蛋白已被成功地分析，以证明这种方法作为现有方法的替代品的有用性。
A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.