O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic Labeling, Copper-Free Click Chemistry, Reductive Cleavage, and Electron-Transfer Dissociation Mass Spectrometry.

As a dynamic post-translational modification, O-linked β-N-acetylglucosamine (O-GlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, O-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for O-GlcNAc enrichment and site mapping. In this method, the O-GlcNAc moiety on peptides is labeled with UDP-GalNAz followed by Cu-free azide-alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides are then released from Neutravidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)trimethyl ammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After being validated by using standard synthetic peptide gCTD and model protein α-crystalline, such an approach was applied to the site mapping of overexpressed TGF-β activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four O-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins.