A New Selective Probe for Visualization and Identification of O‐GlcNAc‐modified Proteins in Cells
Authors: Wang Peng George , Li Jing , Wang Jiajia , Wen Liuqing , Li Shanshan , Zhu He , Ma Cheng , Li Xu 
Published: November 2023

O‐GlcNAcylation (O‐linked N‐acetylglucosamine) of nuclear and cytoplasmic proteins is a ubiquitous and essential glycosylation within multicellular eukaryotes. Metabolic incorporation of chemical probes such as GlcNAc or GalNAc monosaccharide containing an alkyne or azide functional group in conjunction with bioorthogonal reaction provided a powerful tool for detection and identification of O‐GlcNAcylation on cellular proteins. However, these chemical probes also enter into multiple metabolic pathways to produce N‐, O‐glycans and glycolipids, which makes a high background for O‐GlcNAcylation visualization and enrichment. In addition, inside the cells the O‐GlcNAc on the protein is readily cleaved by O‐GlcNAcase (OGA), resulting in less extents of O‐GlcNAcylation. Here we report the development of a new probe, peracetylated 4‐deoxy N‐azidoacetylglucosamine (Ac34DeoGlcNAz), which can be converted by the GalNAc salvage pathway to UDP‐4‐deoxy‐GlcNAz inside the cell and then be transferred onto proteins by O‐GlcNAc transferase (OGT). Due to absence of 4‐hydroxyl group, UDP‐4‐deoxy‐GlcNAz is much less used in the biosynthesis of N‐, O‐glycoproteins and glycoplipids. Moreover, the O‐4‐deoxy‐GlcNAz on the protein cannot be hydrolyzed by OGA, which greatly enhances the incorporation efficiency of this probe for protein O‐GlycNAcylation. Combined with a biotin bioorthogonal conjugate, Ac34DeoGlcNAz allowed for robust visualization of O‐GlcNAc in living cells and identification of O‐GlcNAc‐modified proteins with LC‐MS/MS. This approach represents a more potent and specific strategy to track, capture, and identify O‐GlcNAcylation on proteins in cells and cell lysates. O‐GlcNAcylation (O‐linked N‐acetylglucosamine) of nuclear and cytoplasmic proteins is a ubiquitous and essential glycosylation within multicellular eukaryotes. Metabolic incorporation of chemical probes such as GlcNAc or GalNAc monosaccharide containing an alkyne or azide functional group in conjunction with bioorthogonal reaction provided a powerful tool for detection and identification of O‐GlcNAcylation on cellular proteins. However, these chemical probes also enter into multiple metabolic pathways to produce N‐, O‐glycans and glycolipids, which makes a high background for O‐GlcNAcylation visualization and enrichment. In addition, inside the cells the O‐GlcNAc on the protein is readily cleaved by O‐GlcNAcase (OGA), resulting in less extents of O‐GlcNAcylation. Here we report the development of a new probe, peracetylated 4‐deoxy N‐azidoacetylglucosamine (Ac34DeoGlcNAz), which can be converted by the GalNAc salvage pathway to UDP‐4‐deoxy‐GlcNAz inside the cell and then be transferred onto proteins by O‐GlcNAc transferase (OGT). Due to absence of 4‐hydroxyl group, UDP‐4‐deoxy‐GlcNAz is much less used in the biosynthesis of N‐, O‐glycoproteins and glycoplipids. Moreover, the O‐4‐deoxy‐GlcNAz on the protein cannot be hydrolyzed by OGA, which greatly enhances the incorporation efficiency of this probe for protein O‐GlycNAcylation. Combined with a biotin bioorthogonal conjugate, Ac34DeoGlcNAz allowed for robust visualization of O‐GlcNAc in living cells and identification of O‐GlcNAc‐modified proteins with LC‐MS/MS. This approach represents a more potent and specific strategy to track, capture, and identify O‐GlcNAcylation on proteins in cells and cell lysates.Support or Funding InformationThis work was supported by NIH grant R01GM085267 to P.G.W.