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Mol Cell Proteomics. 2010 Jan;9(1):153-60. doi: 10.1074/mcp.M900268-MCP200. Epub 2009 Aug 19.

Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry.

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Department of Biological Chemistry, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.


Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked beta-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.

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