Abstract
O-Linked β-N-acetylglucosamine (O-GlcNAc) modification found on the serine and threonine residues of intracellular proteins is an inducible post-translational modification that regulates numerous biological processes. In combination with other cell biological and biochemical approaches, a robust and streamlined strategy for detecting the number and stoichiometry of O-GlcNAc modification can provide valuable insights for decoding the functions of O-GlcNAc at the molecular level. Here, we report an optimized workflow for evaluating the O-GlcNAc status of proteins using a combination of metabolic labeling and click chemistry-based mass tagging. This method is strategically complementary to the chemoenzymatic-based mass-tagging method.