Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins
| Autor: | Courtenay R. Hart, Suzanne B. Buck, Jessica F. Dweck, Linda C. Hsieh-Wilson, Peter M. Clark, Daniel E. Mason, Brian J. Agnew, Eric C. Peters |
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| Jazyk: | angličtina |
| Rok vydání: | 2008 |
| Předmět: |
Proteomics
Glycosylation Biotin Biochemistry Catalysis Article Fluorescence Acetylglucosamine chemistry.chemical_compound Colloid and Surface Chemistry Animals Humans Cells Cultured Fluorescent Dyes chemistry.chemical_classification Galactosyltransferase Neurons Cellular imaging Proteins General Chemistry Galactosyltransferases Rats carbohydrates (lipids) Enzyme chemistry Glycoprotein HeLa Cells |
| Popis: | We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide−alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc. |
| Databáze: | OpenAIRE |
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