SRP198469 PRJNA543040 GSE131246 Gene expression analysis of R1 mESCs stably expressing WT ESRRB and Ser 25A (the O-GlcNAc residue mutant) ESRRB Unnatural monosaccharides such as azidosugars that can be metabolically incorporated into cellular glycans are currently used as a major tool for glycan imaging and glycoproteomic profiling. As a common practice to enhance membrane permeability and cellular uptake, the unnatural sugars are per O-acetylated, which, however, can induce a long-overlooked side reaction, non enzymatic S-glycosylation. Herein, we develop 1,3-di-esterified N azidoacetylgalactosamine (GalNAz) as the next generation chemical reporters for metabolic glycan labeling. Both 1,3-di-O-acetylated GalNAz (1,3-Ac2GalNAz) and 1,3-di-O propionylated GalNAz (1,3-Pr2GalNAz) exhibited high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying 1,3-Pr2GalNAz in mouse embryonic stem cells (mESCs), we identified ESRRB, a critical transcription factor for pluripotency, as an O-GlcNAcylated protein. We showed that ESRRB O-GlcNAcylation is important for mESC self-renewal and pluripotency. Mechanistically, ESRRB is O-GlcNAcylated by O-GlcNAc transferase (OGT) at serine 25 (Ser 25), which stabilizes ESRRB, promotes its transcription activity and facilitates its interactions with two master pluripotency regulators, OCT4 and NANOG. Overall design: Three biological replicates of R1 embryonic stem cells stably expressing WT ESRRB and Ser 25A ESRRB were analysed. GSE131246 pubmed 31492838