Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry

Zihao Wang, Namrata D. Udeshi, Meaghan O'Malley, Jeffrey Shabanowitz, Donald F. Hunt, Gerald Warren Hart

Research output: Contribution to journalArticlepeer-review

Abstract

Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked β-N-acetyl-glucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.

Original languageEnglish (US)
Pages (from-to)153-160
Number of pages8
JournalMolecular and Cellular Proteomics
Volume9
Issue number1
DOIs
StatePublished - Jan 2010

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

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