Analysis of alpha-1-acid glycoprotein isoforms using CE-LIF with fluorescent thiol derivatization.

Raúl Garrido-Medina, Angel Puerta, Zuly Rivera-Monroy, Mercedes de Frutos, Andras Guttman, José Carlos Diez-Masa

Index: Electrophoresis 33(7) , 1113-9, (2012)

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Abstract

The analysis of glycoprotein isoforms is of high interest in the biomedical field and clinical chemistry. Many studies have demonstrated that some glycoprotein isoforms could serve as biomarkers for several major diseases, such as cancers and vascular diseases, among others. Capillary zone electrophoresis (CZE) is a well-established technique to separate glycoprotein isoforms, however, it suffers from limited sensitivity when UV-Vis detection is used. On the other hand, with laser-induced fluorescence (LIF) detection, derivatization reaction to render the proteins fluorescent can destroy the resolution of the isoforms. In this work, a derivatization procedure through the thiol groups of glycoproteins using either 5-(iodoacetamide) fluorescein (5-IAF) or BODIPY iodoacetamide is presented with the model protein of alpha-1-acid glycoprotein (AGP). The derivatization process presented enabled high-resolution analysis of AGP isoforms by CZE-LIF. The derivatization procedure was successfully applied to label AGP from samples of serum and secretome of artery tissue, enabling the separation of the AGP isoforms by CE-LIF in natural samples at different concentration levels.© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.