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