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 journalArticle

37 Citations (Scopus)

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

Fingerprint

Alkynes
Chromatography
Purification
Mass spectrometry
Mass Spectrometry
Phosphates
Peptides
alpha-Crystallins
Azides
HeLa Cells
Brain
Proteins
titanium dioxide
Electrons
Tissue

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)

Cite this

Purification and identification of O-GlcNAc-modified peptides using phosphate-based alkyne CLICK chemistry in combination with titanium dioxide chromatography and mass spectrometry. / Parker, Benjamin L.; Gupta, Pankaj; Cordwell, Stuart J.; Larsen, Martin R.; Palmisano, Giuseppe.

In: Journal of Proteome Research, Vol. 10, No. 4, 01.04.2011, p. 1449-1458.

Research output: Contribution to journalArticle

Parker, Benjamin L. ; Gupta, Pankaj ; Cordwell, Stuart J. ; Larsen, Martin R. ; Palmisano, Giuseppe. / Purification and identification of O-GlcNAc-modified peptides using phosphate-based alkyne CLICK chemistry in combination with titanium dioxide chromatography and mass spectrometry. In: Journal of Proteome Research. 2011 ; Vol. 10, No. 4. pp. 1449-1458.
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