A Reversible Chemoenzymatic Labeling Strategy for Profiling of Protein O‐Glucosylation

O‐linked glucose (O‐Glc), the β‐linked modification of serine residues, is a rare form of protein glycosylation first identified on proteins containing epidermal growth factor (EGF)‐like domains (canonical O‐Glc). Several recent studies revealed that proteins lacking EGF‐like domains could also undergo O‐Glc modification (noncanonical O‐Glc). However, the biosynthetic origin and biological function of protein O‐glucosylation remain poorly understood and debated, owing to the lack of effective analytical tools. Here, a reversible chemoenzymatic labeling strategy for O‐Glc analysis is described. By the strategy described, a large number of canonical and noncanonical O‐Glc sites were identified in human cell lines, indicating that protein O‐Glc is a widespread post‐translational protein modification.