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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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journal contribution
posted on 01.04.2011, 00:00 by Benjamin L. Parker, Pankaj Gupta, Stuart J. Cordwell, Martin R. Larsen, Giuseppe Palmisano
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-GlcNAcGalNAzPO4-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.