Charlwood J, Skehel J M, Camilleri P
SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.
Proteomics. 2001 Feb;1(2):275-84. doi: 10.1002/1615-9861(200102)1:2<275::AID-PROT275>3.0.CO;2-K.
Human IgG and IgM, bovine IgM and three therapeutic IgG monoclonal antibodies have been separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Carbohydrates were then released from these immobilised proteins by direct enzymatic digestion, derivatised with a highly fluorescent probe and analysed by high performance liquid chromatography and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. This procedure not only allowed measurement of the purity of the intact antibodies but also provided detailed analysis of the complex mixtures of oligosaccharides covalently attached to these glycoproteins. The methodology out-lined allows the simultaneous processing of a number of glycoproteins separated on one single gel. In contrast to the release of carbohydrate from glycoproteins in solution, this procedure can also be conveniently applied when only impure glycoprotein is available.
人IgG和IgM、牛IgM以及三种治疗性IgG单克隆抗体已通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳进行分离。然后通过直接酶解从这些固定化蛋白质中释放出碳水化合物，用高荧光探针进行衍生化，并通过高效液相色谱和基质辅助激光解吸/电离飞行时间质谱进行分析。该方法不仅能够测定完整抗体的纯度，还能对共价连接到这些糖蛋白上的寡糖复杂混合物进行详细分析。所述方法允许同时处理在一块凝胶上分离的多种糖蛋白。与从溶液中的糖蛋白释放碳水化合物不同，当只有不纯的糖蛋白可用时，该方法也可方便地应用。
