Highly efficient identification of nucleocytoplasmic O‐glycosylation by the TurboID‐based proximity labeling method in living cells

Abstract Glycosylation is a ubiquitous posttranslational modification and plays an important role in many processes, such as protein stability, folding, processing, and trafficking. Among glycosylation types, O‐glycosylation is difficult to analyze due to the complex glycan composition, low abundance and lack of glycosidases to remove the O‐glycans. Many methods have been applied to analyze the O‐glycosylation of membrane glycoproteins and secreted glycoproteins since the synthesis of O‐glycosylation occurred in the Golgi apparatus. In recent years, some O‐glycosylation has been reported in the nucleus. In this work, we present a proximity labeling strategy based on TurboID by combining core 1 β1‐3 galactosyltransferase (C1GalT1), which has been reported in the nucleus, to characterize nucleocytoplasmic O‐glycosylation in living HeLa cells. The O‐glycosylated protein C1GalT1 was biotinylated by the proximity labeling method in living HeLa cells overexpressing C1GalT1 fused by TurboID and enriched by streptavidin‐coated beads. Following digestion with trypsin and mass spectrometry analysis, 68 high‐confidence and 298 putative O‐glycosylated sites were identified on 366 peptides mapped to 267 proteins. These results indicated that the proximity labeling method is a highly efficient technique to identify O‐glycosylation. Furthermore, the finding of abundant O‐glycosylation from nucleocytoplasmic proteins indicates a new pathway of O‐glycosylation synthesis in cells.