Gennaro Lynn A, Delaney Jeannine, Vouros Paul, Harvey David J, Domon Bruno
Department of Chemistry and Barnett Institute, Northeastern University, Boston, MA 02115, USA.
Rapid Commun Mass Spectrom. 2002;16(3):192-200. doi: 10.1002/rcm.564.
The increasing interest in the development of glycoproteins for therapeutic purposes has created a greater demand for methods to characterize the sugar moieties bound to them. Traditionally, released carbohydrates are derivatized using such methods as permethylation or fluorescent tagging prior to analysis by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), or direct infusion mass spectrometry. However, little research has been performed using CE with on-line mass spectrometry (MS) detection. The CE separation of neutral oligosaccharides requires the covalent attachment of a charged species for electrophoretic migration. Among charged labels which have shown promise in assisting CE and HPLC separation is the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). This report describes the qualitative profiling of charged ANTS-derivatized and underivatized complex glycans by CE with on-line electrospray ion trap mass spectrometry. Several neutral standard glycans including a maltooligosaccharide ladder were derivatized with ANTS and subjected to CE/UV and CE/MS using low pH buffers consisting of citric and 6-aminocaproic acid salts. The ANTS-derivatized species were detected as negative ions, and multiple stage MS analysis provided valuable structural information. Fragment ions were easily identified, showing promise for the identification of unknowns. N-Linked glycans released from bovine fetuin were used to demonstrate the applicability of ANTS derivatization followed by CE/MS for the analysis of negatively charged glycans. Analyses were performed on both underivatized and ANTS-derivatized species, and sialylated glycans were separated and detected in both forms. The ability of the ion trap mass spectrometer to perform multiple stage analysis was exploited, with MS5 information obtained on selected glycans. This technique presents a complementary method to existing methodologies for the profiling of glycan mixtures.
出于治疗目的对糖蛋白开发的兴趣日益增加，这使得对表征与之结合的糖部分的方法有了更大的需求。传统上，在通过高效液相色谱（HPLC）、毛细管电泳（CE）或直接进样质谱分析之前，使用诸如全甲基化或荧光标记等方法对释放的碳水化合物进行衍生化。然而，使用CE与在线质谱（MS）检测进行的研究很少。中性寡糖的CE分离需要共价连接一种带电物质以进行电泳迁移。在已显示出有助于CE和HPLC分离的带电标记物中，荧光团8-氨基萘-1,3,6-三磺酸（ANTS）是其中之一。本报告描述了通过CE与在线电喷雾离子阱质谱对带电的ANTS衍生化和未衍生化的复合聚糖进行定性分析。几种中性标准聚糖，包括麦芽寡糖阶梯，用ANTS进行衍生化，并使用由柠檬酸和6-氨基己酸盐组成的低pH缓冲液进行CE/UV和CE/MS分析。ANTS衍生化的物质被检测为负离子，多级MS分析提供了有价值的结构信息。碎片离子很容易识别，显示出鉴定未知物的前景。从牛胎球蛋白释放的N-连接聚糖被用于证明ANTS衍生化后接着进行CE/MS用于分析带负电荷聚糖的适用性。对未衍生化和ANTS衍生化的物质都进行了分析，并且唾液酸化聚糖以两种形式都被分离和检测到。利用了离子阱质谱仪进行多级分析的能力，获得了选定聚糖的MS5信息。该技术为聚糖混合物分析的现有方法提供了一种补充方法。
