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Mapping Sites of O-GlcNAc Modification Using Affinity Tags for Serine and Threonine Post-translational Modifications*

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Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-Linked β-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic modification more analogous to phosphorylation than to classical complex O-glycosylation. We describe a mass spectrometry-based method for the identification of sites modified by O-GlcNAc that relies on mild β-elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace dithiothreitol as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites can be mapped using tandem mass spectrometry. This same methodology can be applied to mapping sites of serine and threonine phosphorylation, and we provide a strategy that uses modification-specific antibodies and enzymes to discriminate between the two post-translational modifications. The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin I purified from rat brain. BEMAD was then used on a purified nuclear pore complex preparation to map novel sites of O-GlcNAc modification on the Lamin B receptor and the nucleoporin Nup155. This method is amenable for performing quantitative mass spectrometry and can also be adapted to quantify cysteine residues. In addition, our studies emphasize the importance of distinguishing between O-phosphate versus O-GlcNAc when mapping sites of serine and threonine post-translational modification using β-elimination/Michael addition methods.

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Published, MCP Papers in Press, September 26, 2002, DOI 10.1074/mcp.M200048-MCP200

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This work was supported by National Institutes of Health Grants CA43486 and DK61671 (to G. W. H.), American Cancer Society Grant RSG-01-064-01-CSM (to M. J. M.), and National Research Service Award Fellowships CA83261 (to L. W.) and GM20528 (to K. V.). MALDI analysis was performed in the Johns Hopkins School of Medicine, Applied Biosystems Mass Spectrometry facility, which is funded in part by the Johns Hopkins Fund for Medical Discovery, the Institute for Cell Engineering, and National Center for Research Resources Shared Instrumentation Grant 1S10-RR14702 (to G. W. H.). Under a licensing agreement between Covance Research Products, Hoffman LaRoche, and The Johns Hopkins University, Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody. The terms of this arrangement are being managed by The Johns Hopkins University in accordance with its conflict of interest policies.

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Both authors contributed equally to this work.