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.