Journal
JOURNAL OF PROTEOME RESEARCH
Volume 10, Issue 4, Pages 1449-1458Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr100565j
Keywords
O-GlcNAc; phosphorylation; HCD MS/MS; ETD MS/MS; chemoenzymatic labeling; titanium dioxide chromatography; post-translational modifications
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Funding
- Lundbeck Foundation
- Danish Natural Science Research Council [MRL 09-06-S989]
- National Health and Medical Research Council (NHMRC) [SJC 571002]
- University of Sydney
- Faculty of Medicine
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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-GlcNAcGaINAzPO(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 alphacrystallin 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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