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Updated: Jun 6, 2026
A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues
Published on: May 4, 2022
Purification and identification of O-GlcNAc-modified peptides using phosphate-based alkyne CLICK chemistry in
Benjamin L Parker1, Pankaj Gupta, Stuart J Cordwell
1Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
Abstract:
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.
