title: Simple and specific dual-wavelength excitable dye staining for glycoprotein detection in polyacrylamide gels and its application in glycoproteomics.
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.
authors: Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.
authors: Yu-Hsuan
authors: YH
authors: Chiang
authors: Yu-Jen
authors: YJ
authors: Wu
authors: Ya-Ting
authors: YT
authors: Lu
authors: Kuan-Hung
authors: KH
authors: Chen
authors: Tzu-Chun
authors: TC
authors: Lin
authors: Yu-Kuang H
authors: YK
authors: Chen
authors: Ding-Tzai
authors: DT
authors: Li
authors: Fong-Ku
authors: FK
authors: Shi
authors: Ching-Chuan
authors: CC
authors: Chen
authors: Jue-Liang
authors: JL
authors: Hsu
mesh_terms: D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry* / Q000652:urine; D006801:Humans; D010446:Peptide Fragments; D011247:Pregnancy; D040901:Proteomics / Q000379:methods*; D012235:Rhodamines / Q000737:chemistry*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D005456:Fluorescent Dyes; D006023:Glycoproteins; D010446:Peptide Fragments; D012235:Rhodamines; C022027:lissamine rhodamine B