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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.

SUBMITTER: Clark PM

PROVIDER: S-EPMC2649877 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins.

Clark Peter M PM Dweck Jessica F JF Mason Daniel E DE Hart Courtenay R CR Buck Suzanne B SB Peters Eric C EC Agnew Brian J BJ Hsieh-Wilson Linda C LC

Journal of the American Chemical Society 20080807 35

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 ...[more]

PMID: 18683930

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