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Journal of Biomedicine and Biotechnology
Volume 2011 (2011), Article ID 780108, 8 pages
http://dx.doi.org/10.1155/2011/780108
Methodology Report

Simple and Specific Dual-Wavelength Excitable Dye Staining for Glycoprotein Detection in Polyacrylamide Gels and Its Application in Glycoproteomics

1Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan
2Department of Beauty Science, Meiho University of Technology, Pingtung 91202, Taiwan
3Mass Solutions Technology Co. Ltd., New Taipei City 22101, Taiwan
4Food Science and Nutrition Department, Meiho University of Science and Technology, Pingtung 91202, Taiwan
5Department of Plant Industry, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan

Received 20 May 2011; Accepted 4 August 2011

Academic Editor: Susan A. Rotenberg

Copyright © 2011 Yu-Hsuan Chiang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

In this study, a commercially available fluorescent dye, Lissamine rhodamine B sulfonyl hydrazine (LRSH), was designed to specifically stain the glycoproteins in polyacrylamide gels. Through the periodate/Schiff base mechanism, the fluorescent dye readily attaches to glycoproteins and the fluorescence can be simultaneously observed under either 305 nm or 532 nm excitation therefore, the dye-stained glycoproteins can be detected under a regular UV transilluminator or a more elegant laser-based gel scanner. The specificity and detection limit were examined using a standard protein mixture in polyacrylamide gels in this study. The application of this glycoprotein stain dye was further demonstrated using pregnancy urine samples. The fluorescent spots were further digested in gel and their identities confirmed through LC-MS/MS analysis and database searching. In addition, the N-glycosylation sites of LRSH-labeled uromodulin were readily mapped via in-gel PNGaseF deglycosylation and LC-MS/MS analysis, which indicated that this fluorescent dye labeling does not interfere with enzymatic deglycosylation. Hence, the application of this simple and specific dual-wavelength excitable dye staining in current glycoproteome research is promising.