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Submitted on August 20, 2002
Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, MD 21205
Corresponding Author: gwhart{at}jhmi.edu
Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-linked
Revised on September 23, 2002
Accepted on September 26, 2002
Mapping Sites of O-GlcNAc Modification Using Affinity Tags for Serine and Threonine Post-Translational Modifications
-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 -elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace DTT as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites 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 (NPC) 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 b-elimination/Michael addition methods.
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