Chemoproteomic Profiling of O‐GlcNAcylation in Arabidopsis Thaliana by Using Metabolic Glycan Labeling

Protein O‐GlcNAcylation is a ubiquitous posttranslational modification occurring both in animals and plants. While thousands of O‐GlcNAcylated proteins have been identified in animals, the plant O‐GlcNAcylated proteome remains poorly studied. Herein we report the development of a chemoproteomic strategy for profiling of O‐GlcNAcylated proteins in Arabidopsis based on the metabolic glycan labeling (MGL) method. We first demonstrated that both N‐azidoacetylglucosamine (GlcNAz) and N‐azidoacetylgalactosamine (GalNAz) can metabolically label O‐GlcNAc with azides in Arabidopsis seedlings. Arabidopsis UDP‐galactose 4‐epimerases were found to interconvert UDP‐GalNAz and UDP‐GlcNAz, supporting the existence of a GalNAc metabolism pathway. By tagging the azide‐incorporated O‐GlcNAc with alkyne‐biotin via click chemistry, the O‐GlcNAcylated proteins were enriched and analyzed by mass spectrometry. We identified 645 candidate O‐GlcNAcylated proteins in Arabidopsis seedlings, of which 592 were newly identified. The identified O‐GlcNAcylated proteins were enriched in various plant‐specific processes such as hormone responses. By co‐expression of a selected list of the identified proteins with SECRET AGENT, the Arabidopsis O‐GlcNAc transferase, we validated that the MGL‐identified proteins were O‐GlcNAc‐modified. Our work establishes a powerful tool for profiling plant O‐GlcNAylation and provides an invaluable resource for investigating the functional role of O‐GlcNAc in Arabidopsis.