Mapping Sites of O-GlcNAc Modification Using Affinity Tags for Serine and Threonine Post-translational Modifications

TLDR

Identifying sites of post‑translational modifications on proteins is a major challenge in proteomics, and O‑GlcNAc is a dynamic nucleocytoplasmic modification analogous to phosphorylation. The authors present a mass‑spectrometry method that uses mild beta‑elimination followed by Michael addition with dithiothreitol (BEMAD) to identify O‑GlcNAc sites. Synthetic peptides demonstrate that biotin pentylamine can replace dithiothreitol as the nucleophile; the resulting peptides are enriched by affinity chromatography and mapped by tandem MS, and the approach can be extended to serine/threonine phosphorylation using modification‑specific antibodies and enzymes. BEMAD was validated by mapping six O‑GlcNAc sites on Synapsin I and novel sites on Lamin B receptor and Nup155, and it is adaptable for quantitative MS and cysteine quantification while highlighting the need to distinguish O‑phosphate from O‑GlcNAc.

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.