CHARACTERIZATION OF N-LINKED OLIGOSACCHARIDES BY ELECTROSPRAY AND TANDEM MASS-SPECTROMETRY
CHARACTERIZATION OF N-LINKED OLIGOSACCHARIDES BY ELECTROSPRAY AND TANDEM MASS-SPECTROMETRY
复制标题
DOI：
10.1021/ac00037a024
复制
发表时间：
1992-07-01
影响因子：
7.4
通讯作者：
HENION, JD
中科院分区：
文献类型：
--
作者：
DUFFIN, KL;WELPLY, JK;HENION, JD
电喷雾和串联质谱仪用于表征从糖蛋白的天冬酰胺侧链中消化出来的未衍化低聚糖。在负离子检测模式下，含唾液酸寡糖的检测灵敏度最高，而在正离子检测模式下，不含唾液酸的低聚糖检测灵敏度最高。在样品溶剂中加入10 mM醋酸钠或醋酸铵，可显著提高电喷雾质谱中寡糖的正离子丰度。用串联质谱仪确定了低聚糖的一级结构特征。已发展的方法学已经分支的高甘露糖，杂交和复合碳水化合物标准被应用于低聚糖的混合物，这些低聚糖被N-葡聚糖酶从糖蛋白，卵清蛋白中消化。与阴离子交换层析/脉冲安培检测法和放射性标记低聚糖还原端的凝胶渗透色谱相比，电喷雾质谱法得到的低聚糖的组成和相对丰度要好于阴离子交换层析/脉冲安培检测法。卵清蛋白的寡糖含量独立于完整的44 kDa糖蛋白电喷雾质谱图中观察到的不均一性。对酶消化前后低聚糖组成的比较表明，N-葡聚糖酶选择性地消化高甘露糖和低分子低聚糖。
Electrospray and tandem mass spectrometry are used to characterize underivatized oligosaccharides that have been digested from asparagine side chains of glycoproteins. Oligosaccharides thal contain sialic acids were detected with the best sensitivity in the negative-ion detection mode whereas those that do not contain sialic acid were detected with the best sensitivity in the positive-ion detection mode. The positive-ion abundances of oligosaccharides were greatly enhanced in electrospray mass spectra by adding 10 mM sodium acetate or ammonium acetate to the sample solvent. Tandem mass spectrometry was used to determine primary structural features of the oligosaccharides. Methodology that has been developed on branched high-mannose, hybrid, and complex carbohydrate standards was applied to a mixture of oligosaccharides that were digested with N-glycanase from the glycoprotein, ovalbumin. The composition and relative abundances of individual oligosaccharides obtained from the electrospray mass spectrum compare favorably to those obtained by anion-exchange chromatography/pulsed amperometric detection and by gel permeation chromatography of the oligosaccharides after radiolabeling the reducing end of the carbohydrates. The oligosaccharide content of ovalbumin was independently determined from the heterogeneity observed in the electrospray mass spectrum of the Intact 44-kDa glycoprotein. Comparison of the oligosaccharide compositions determined before and after enzymatic digestion shows a selective digestion of high mannose and low molecular weight oligosaccharides by N-glycanase.