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

Benjamin L. Parker, Pankaj Gupta, Stuart J. Cordwell, Martin R. Larsen, Giuseppe Palmisano

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)1449-1458
Number of pages10
JournalJournal of Proteome Research
Volume10
Issue number4
DOIs
Publication statusPublished - Apr 1 2011

Keywords

  • chemoenzymatic labeling
  • ETD MS/MS
  • HCD MS/MS
  • O-GlcNAc
  • phosphorylation
  • post-translational modifications
  • titanium dioxide chromatography

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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