Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications.

Lance Wells, Keith Vosseller, Robert N Cole, Janet M. Cronshaw, Michael J Matunis, Gerald Warren Hart

Research output: Contribution to journalArticle

Abstract

Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-Linked beta-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic modification more analogous to phosphorylation than to classical complex O-glycosylation. We describe a mass spectrometry-based method for the identification of sites modified by O-GlcNAc that relies on mild beta-elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace dithiothreitol as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites can be mapped using tandem mass spectrometry. This same methodology can be applied to mapping sites of serine and threonine phosphorylation, and we provide a strategy that uses modification-specific antibodies and enzymes to discriminate between the two post-translational modifications. The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin I purified from rat brain. BEMAD was then used on a purified nuclear pore complex preparation to map novel sites of O-GlcNAc modification on the Lamin B receptor and the nucleoporin Nup155. This method is amenable for performing quantitative mass spectrometry and can also be adapted to quantify cysteine residues. In addition, our studies emphasize the importance of distinguishing between O-phosphate versus O-GlcNAc when mapping sites of serine and threonine post-translational modification using beta-elimination/Michael addition methods.

Original languageEnglish (US)
Pages (from-to)791-804
Number of pages14
JournalMolecular and Cellular Proteomics
Volume1
Issue number10
StatePublished - Oct 2002

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Dithiothreitol
Threonine
Post Translational Protein Processing
Serine
Mass spectrometry
Phosphorylation
Mass Spectrometry
Nuclear Pore Complex Proteins
Synapsins
Glycosylation
Affinity chromatography
Nuclear Pore
Peptides
Nucleophiles
Acetylglucosamine
Biotin
Tandem Mass Spectrometry
Affinity Chromatography
Proteomics
Cysteine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. / Wells, Lance; Vosseller, Keith; Cole, Robert N; Cronshaw, Janet M.; Matunis, Michael J; Hart, Gerald Warren.

In: Molecular and Cellular Proteomics, Vol. 1, No. 10, 10.2002, p. 791-804.

Research output: Contribution to journalArticle

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