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

Abstract

<p class="first" id="P5">As a dynamic post-translational modification, O-linked <i>β</i>-N-acetylglucosamine ( <i>O</i>-GlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, <i>O</i>-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for <i>O</i>-GlcNAc enrichment and site mapping. In this method, the <i>O</i>-GlcNAc moiety on peptides was labeled with UDP–GalNAz followed by copper-free azide–alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides were then released from NeutrAvidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)trimethylammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After validation by using standard synthetic peptide gCTD and model protein <i>α</i>-crystallin, such an approach was applied to the site mapping of overexpressed TGF- <i>β</i>-activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four <i>O</i>-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins. </p><p id="P6"> <div class="figure-container so-text-align-c"> <img alt="" class="figure" src="/document_file/eba71850-83a8-4ec1-820e-b76f0ef52fa5/PubMedCentral/image/nihms-1051119-f0001.jpg"/> </div> </p>