Friedman Y, Higgins E A
Genzyme Corporation, Framingham, Massachusetts 01701-9322, USA.
Anal Biochem. 1995 Jul 1;228(2):221-5. doi: 10.1006/abio.1995.1342.
We have developed a method for monitoring the N-glycosylation of recombinant glycoproteins directly from conditioned medium samples. Proteins in the conditioned medium are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes. After staining the membranes with Coomassie blue, the protein(s) of interest is excised. Oligosaccharides are released from the membrane-bound glycoprotein by digesting with peptide N4-(acetyl-beta-glucosaminyl) asparagine amidase and labeled with the fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS). Labeled oligosaccharides are then separated on polyacrylamide gels which allow for the direct comparison of samples. We have shown that recombinant human lysosomal hydrolase alpha-galactosidase A is N-glycosylated with both sialylated and phosphorylated oligosaccharides. ANTS-labeled oligosaccharide bands from alpha-galactosidase A were isolated from polyacrylamide gels. Sialylated and phosphorylated bands were identified by shifts in their electrophoretic mobility after digesting with neuraminidase or alkaline phosphatase to remove sialic acid or phosphate groups, respectively. Using the ANTS-labeled oligosaccharides from alpha-galactosidase A, we have shown that polyacrylamide gels can be used to resolve sialylated and phosphorylated oligosaccharide structures.
我们开发了一种直接从条件培养基样品中监测重组糖蛋白N-糖基化的方法。条件培养基中的蛋白质通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离，然后电印迹到聚偏二氟乙烯膜上。用考马斯亮蓝对膜染色后，切下感兴趣的蛋白质。通过用肽N4-（乙酰-β-氨基葡萄糖基）天冬酰胺酶消化，从膜结合糖蛋白中释放寡糖，并用荧光团8-氨基萘-1,3,6-三磺酸盐（ANTS）标记。然后在聚丙烯酰胺凝胶上分离标记的寡糖，从而可以直接比较样品。我们已经表明，重组人溶酶体水解酶α-半乳糖苷酶A被唾液酸化和磷酸化的寡糖进行N-糖基化。从聚丙烯酰胺凝胶中分离出α-半乳糖苷酶A的ANTS标记的寡糖条带。通过分别用神经氨酸酶或碱性磷酸酶消化以去除唾液酸或磷酸基团后，其电泳迁移率的变化来鉴定唾液酸化和磷酸化的条带。使用来自α-半乳糖苷酶A的ANTS标记的寡糖，我们已经表明聚丙烯酰胺凝胶可用于解析唾液酸化和磷酸化的寡糖结构。
