Discrimination of glycoproteins via two-color laser-induced fluorescence detection coupled with postcolumn derivatization in capillary electrophoresis
Discrimination of glycoproteins via two-color laser-induced fluorescence detection coupled with postcolumn derivatization in capillary electrophoresis
复制标题
通过双色激光诱导荧光检测结合毛细管电泳中的柱后衍生化来区分糖蛋白
DOI：
10.1002/elps.201300149
复制
发表时间：
2013
期刊：
影响因子：
2.9
通讯作者：
Takashi Kaneta
中科院分区：
文献类型：
--
作者：
Ayumi Tabara;Takashi Kaneta
在这里，我们报告了一种新的方法，包括毛细管电泳分离，然后用柱后衍生的双色LIF检测。该方法可用于区分含有糖基化和非糖基化蛋白质的蛋白质混合物中的糖蛋白。检测器允许同时测量分别用萘-2，3-二甲醛和四甲基罗丹明标记的伴刀豆球蛋白A（Con A）（罗丹明标记的Con A）进行柱后衍生后，通过450 nm（二极管激光）和532 nm（Nd：YAG激光）激光激发天然蛋白获得的两个电泳图。因此，如果蛋白质在两个电泳图中在相同的迁移时间处显示峰，则可以将其指定为糖基化的。根据提出的原理，在一次运行中，我们在罗丹明标记的Con A存在下区分了糖基化蛋白（甲状腺球蛋白）和非糖基化蛋白（白蛋白）。因为该方法允许用荧光标记探针同时检测天然蛋白质及其复合物，所以它应该对结合测定具有广泛的适用性。
Here, we report a novel method consisting of capillary electrophoretic separation followed by two‐color LIF detection with postcolumn derivatization. The method can be used to discriminate glycoproteins in a protein mixture containing both glycosylated and unglycosylated proteins. The detector permitted simultaneous measurements of two electropherograms obtained by 450 nm (diode laser) and 532 nm (Nd:YAG laser) lasers excited native proteins following postcolumn derivatization with naphthalene‐2,3‐dicarboxaldehyde and concanavalin A (Con A) labeled with tetramethylrhodamine (rhodamine‐labeled Con A), respectively. So, a protein can be assigned as glycosylated if it shows a peak at the same migration time in both electropherograms. According to the proposed principle, in a single run we discriminated a glycosylated protein (thyroglobulin) from an unglycosylated protein (albumin) in the presence of rhodamine‐labeled Con A. Because the methodology permits the simultaneous detection of native proteins and their complexes with a fluorescently labeled probe, it should have broad applicability to binding assays.