Reversible hydrazide chemistry-based enrichment for O-GlcNAc-modified peptides and glycopeptides having non-reducing GlcNAc residues

O-Linked N-acetylglucosamine (O-GlcNAc) is an emerging post-translational modification (PTM) of proteins. Analysis of O-GlcNAc modification using mass spectrometry (MS) is often problematic because of the low stoichiometry of the modification. In this study, we developed a new method for enriching O-GlcNAc-modified peptides using reversible hydrazide chemistry. O-GlcNAc-modified peptides were first labeled with N-azidoacetylgalactosamine (GalNAz) using gatactosyltransferase-T1 (Y289L) enzyme. The azide group on the GalNAz residue was then reacted with 3-ethynylbenzaldehyde via coppercatalyzed Huisgen 1,3-cycloaddition click reactionto form an aromatic aldehyde group of glycopeptides. Aromatic aldehyde-derivatized glycopeptides were enriched by reversible hydrazone formation with hydrazide resin. Reaction conditions for each step, especially for the click reaction, were optimized to achieve complete reaction without significant side reactions. This method was validated using a tryptic digest of bovine a-crystallin, which is an O-GlcNAc-modified glycoprotein. The developed method was also applied to structure-specific enrichment of N-linked glycopeptides having nonreducing terminal GlcNAc residues. All materials and chemicals required for this method are commercially available and there is no need to prepare any special reagents, facilitating the introduction of this method in any laboratory.