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J Proteome Res. 2011 Apr 1;10(4):1449-58. doi: 10.1021/pr100565j. Epub 2011 Feb 22.

Purification and identification of O-GlcNAc-modified peptides using phosphate-based alkyne CLICK chemistry in combination with titanium dioxide chromatography and mass spectrometry.

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Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.


A selective method for the enrichment of O-GlcNAcylated peptides using a novel CLICK chemistry reagent is described. Peptides modified by O-GlcNAc were enzymatically labeled with N-azidoacetylgalactosamine. The azide was then reacted with a phospho-alkyne using CLICK chemistry and O-GlcNAcGalNAzPO(4)-containing peptides were enriched using titanium dioxide chromatography. Modified peptides were analyzed using a combination of higher energy collision dissociation for identification and electron transfer dissociation to localize the site of O-GlcNAc attachment. The enrichment method was developed and optimized using an alpha-crystallin standard protein and then applied to a soluble protein preparation of mouse brain tissue and a nuclear preparation generated from HeLa cells. A total of 42 unique O-GlcNAcylated peptides were identified, including 7 novel O-GlcNAc sites.

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