Lemoine J, Cabanes-Macheteau M, Bardor M, Michalski J C, Faye L, Lerouge P
Laboratoire de Chimie Biologique, CNRS UMR 8576, Universit¿e de Lille, 59655 Villeneuve d'Ascq, France.
Rapid Commun Mass Spectrom. 2000;14(2):100-4. doi: 10.1002/(SICI)1097-0231(20000130)14:2<100::AID-RCM845>3.0.CO;2-W.
Fluorophore-assisted carbohydrate electrophoresis (FACE) is a fast and efficient analytical method which is now widely used in glycobiology for the separation and quantification of free or glycoprotein-released oligosaccharides. However, since identification by FACE of N-glycan structures is only based on their electrophoretic mobility after labelling with 8-aminonaphthalene-1,3, 6-trisulfonic acid (ANTS), co-migration of derived glycans on gel could occur which may result in erroneous structural assignments. As a consequence, a protocol was developed for the fast and efficient matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometric analysis of ANTS-labelled N-glycans. N-Glycans were isolated from plant and mammalian glycoproteins, reductively aminated with the charged fluorophore 8-aminonaphthalene-1, 3, 6-trisulfonic acid (ANTS) and separated using high resolution polyacrylamide gel electrophoresis. The ANTS-labelled glycans were eluted from FACE gel slices and then analysed by MALDI-TOF mass spectrometry in negative ion mode. Using 3-aminoquinoline containing 2.5 mM citrate NH(4)(+) as matrix, neutral N-linked N-glycans, as well as labelled sialylated oligosaccharides, were found to be easily detected in the 2-10 picomole range giving rise to ¿M - H(-) ions.
荧光团辅助碳水化合物电泳（FACE）是一种快速高效的分析方法，目前在糖生物学中广泛用于游离或糖蛋白释放的寡糖的分离和定量。然而，由于通过FACE鉴定N-聚糖结构仅基于其用8-氨基萘-1,3,6-三磺酸（ANTS）标记后的电泳迁移率，凝胶上衍生聚糖可能会发生共迁移，这可能导致错误的结构归属。因此，开发了一种用于ANTS标记的N-聚糖的快速高效基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱分析的方案。从植物和哺乳动物糖蛋白中分离出N-聚糖，用带电荷的荧光团8-氨基萘-1,3,6-三磺酸（ANTS）进行还原胺化，并用高分辨率聚丙烯酰胺凝胶电泳进行分离。从FACE凝胶切片中洗脱ANTS标记的聚糖，然后在负离子模式下通过MALDI-TOF质谱分析。使用含有2.5 mM柠檬酸铵（NH₄⁺）的3-氨基喹啉作为基质，发现中性N-连接的N-聚糖以及标记的唾液酸化寡糖在2-10皮摩尔范围内易于检测到，产生[M - H]⁻离子。
