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O-GlcNAcylation is the only sugar modification for proteins present in the cytoplasm and nucleus and is thought to be involved in the regulation of protein function and localization. Currently, several methods are known for detecting O-GlcNAcylated proteins using monoclonal antibodies or wheat germ agglutinin, but these methods have some limitations in their sensitivity and quantitative comparison. We developed a new disaccharide-tag method to overcome these problems. This is a method in which a soluble GalNAc transferase is expressed intracellularly, extended to a disaccharide of GalNAc-GlcNAc, and detected using a Wisteria japonica agglutinin specific to this disaccharide. We verified the method using human c-Rel protein and also highly sensitively compared the difference in O-GlcNAc modification of intracellular proteins associated with differentiation from embryonic stem cell (ESC) to epiblast-like cells (EpiLC). As one example of such a modification, a novel O-GlcNAc modification was found in the transcription factor Sox2 at residue Ser263, and the modification site could be identified by nano liquid chromatography-mass spectrometry.

Embryonic stem cells (ESCs) and epiblast-like cells (EpiLCs) recapitulate in vitro the epiblast first cell lineage decision, allowing characterization of the molecular mechanisms underlying pluripotent state transition. Here, we performed a comprehensive and comparative analysis of total glycomes of mouse ESCs and EpiLCs, revealing that overall glycosylation undergoes dramatic changes from early stages of development. Remarkably, we showed for the first time the presence of a developmentally regulated network orchestrating glycosylation changes and identified polycomb repressive complex 2 (PRC2) as a key component involved in this process. Collectively, our findings provide novel insights into the naïve-to-primed pluripotent state transition and advance the understanding of glycosylation complex regulation during early mouse embryonic development.