Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins
Abstract
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.
Additional Information
© 2008 American Chemical Society. Received April 24, 2008. Publication Date (Web): August 7, 2008. We thank Dr. P. Qasba for providing the mutant GalT plasmid, J. Rexach for providing neurons and helpful discussions, T. Nyberg for helpful discussions, and Prof. N. Pierce for use of the FujiFilm imager. This work was supported by the NIH (RO1 GM084724) and Howard Hughes Medical Institute.Attached Files
Accepted Version - nihms90912.pdf
Supplemental Material - ja8030467_si_001.pdf
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Additional details
- PMCID
- PMC2649877
- Eprint ID
- 57671
- DOI
- 10.1021/ja8030467
- Resolver ID
- CaltechAUTHORS:20150519-144346544
- RO1 GM084724-05
- NIH
- Howard Hughes Medical Institute (HHMI)
- Created
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2015-05-19Created from EPrint's datestamp field
- Updated
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2021-11-10Created from EPrint's last_modified field