Gennaro Lynn A, Harvey David J, Vouros Paul
Department of Chemistry and Barnett Institute, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
Rapid Commun Mass Spectrom. 2003;17(14):1528-34. doi: 10.1002/rcm.1079.
The significant complexity, similar polarity and lack of ionizable sites make the analysis of glycans an analytical challenge. These compounds are often derivatized and separated by normal-phase high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) followed by UV or fluorescence detection. Due to widespread use of reversed-phase chromatography coupled to electrospray mass spectrometry as an analytical tool, our laboratory has developed this methodology for the analysis of glycans derivatized with a negatively charged tag, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). It is possible to exploit the ability of this negatively charged tag to interact with a mobile phase ion-pairing reagent, allowing retention on a reversed-phase C(18) column for subsequent on-line UV or MS analysis. ANTS-derivatized samples, including a maltooligosaccharide ladder and glycans released from bovine ribonuclease B, bovine fetuin, and chicken ovalbumin, were analyzed using this method. In addition to reversed-phase retention, ribonuclease B and ovalbumin derivatives displayed highly desirable isomeric separation. With the use of mass spectrometric detection for glycan identity, this allowed relative quantitation of individual components.
聚糖结构的显著复杂性、相似的极性以及缺乏可电离位点使得聚糖分析成为一项分析挑战。这些化合物通常先进行衍生化处理，然后通过正相高效液相色谱（HPLC）或毛细管电泳（CE）进行分离，随后采用紫外或荧光检测。鉴于反相色谱与电喷雾质谱联用作为一种分析工具的广泛应用，我们实验室开发了这种方法，用于分析用带负电荷的标签8-氨基萘-1,3,6-三磺酸（ANTS）衍生化的聚糖。利用这种带负电荷的标签与流动相离子对试剂相互作用的能力，可使其保留在反相C(18)柱上，以便随后进行在线紫外或质谱分析。使用该方法分析了ANTS衍生化的样品，包括麦芽寡糖阶梯以及从牛核糖核酸酶B、牛胎球蛋白和鸡卵清蛋白释放的聚糖。除了反相保留外，核糖核酸酶B和卵清蛋白衍生物还显示出非常理想的异构体分离效果。通过使用质谱检测来确定聚糖的身份，这使得能够对各个组分进行相对定量分析。
