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Discrimination of glycoproteins via two‐color laser‐induced fluorescence detection coupled with postcolumn derivatization in capillary electrophoresis
Author(s) -
Tabara Ayumi,
Kaneta Takashi
Publication year - 2013
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201300149
Subject(s) - electropherogram , chemistry , chromatography , capillary electrophoresis , derivatization , fluorescence , electrophoresis , glycoprotein , laser induced fluorescence , rhodamine , mass spectrometry , biochemistry , physics , quantum mechanics
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.