Abstract
Mechanistic studies of O-GlcNAc glycosylation have been limited by an inability to monitor the glycosylation stoichiometries of proteins obtained from cells. Here we describe a powerful method to visualize the O-GlcNAc–modified protein subpopulation using resolvable polyethylene glycol mass tags. This approach enables rapid quantification of in vivo glycosylation levels on endogenous proteins without the need for protein purification, advanced instrumentation or expensive radiolabels. In addition, it establishes the glycosylation state (for example, mono-, di-, tri-) of proteins, providing information regarding overall O-GlcNAc site occupancy that cannot be obtained using mass spectrometry. Finally, we apply this strategy to rapidly assess the complex interplay between glycosylation and phosphorylation and discover an unexpected reverse 'yin-yang' relationship on the transcriptional repressor MeCP2 that was undetectable by traditional methods. We anticipate that this mass-tagging strategy will advance our understanding of O-GlcNAc glycosylation, as well as other post-translational modifications and poorly understood glycosylation motifs.
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Acknowledgements
We thank the following people for their generous gifts: G. Hart (The Johns Hopkins University School of Medicine) for the OGT antibody, S. Whiteheart (University of Kentucky College of Medicine) for the OGA antibody, W. Wang and K. Hu (US National Institute of Aging) for the MeCP2 antibodies, P. Greengard and A. Nairn (The Rockefeller University) for the synapsin antibody and P. Qasba (US National Cancer Institute) for the Y289L GalT construct. We thank P. Clark and T. Wilson for a critical reading of the manuscript. We also thank P. Clark (California Institute of Technology) for preparing the Y289L GalT used in these studies and for contributing to the aminooxy-linker synthesis. This work was supported by grants from the US National Institutes of Health (R01 GM084724 to L.C.H.-W. and F31 NS056525-02 to J.E.R.) and the Rett Syndrome Research Foundation (Y.E.S.).
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J.E.R. and L.C.H.-W. conceived of and designed the experiments. J.E.R. performed the biochemistry and labeling experiments, J.T. and Y.E.S. contributed to the development and characterization of the phospho-specific antibody, C.J.R. synthesized and characterized the first set of aminooxy-functionalized PEG probes and S.-H.Y. synthesized the UDP-ketogalactose substrate and additional aminooxy-functionalized PEG probes. J.E.R. and L.C.H.-W. wrote the manuscript, and all authors participated in editing the manuscript.
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Rexach, J., Rogers, C., Yu, SH. et al. Quantification of O-glycosylation stoichiometry and dynamics using resolvable mass tags. Nat Chem Biol 6, 645–651 (2010). https://doi.org/10.1038/nchembio.412
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DOI: https://doi.org/10.1038/nchembio.412
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