iProXProteomicsYanting SuHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX00036750002014202040134@whu.edu.cnResearch Center of Basic Medical Sciences, School of Basic Medical Sciences, Hubei University of Science and TechnologyiProXTurboID, a proximity labelling method based on mutant biotin ligase, is an efficient new technique for recognizing protein-protein interactions and has been successfully applied to living cells. Sialic acid is typically the terminal monosaccharide attached to many glycoproteins and plays many important roles in many biological processes. However, the lack of enrichment methods for terminal sialic acid glycosylation <i>in vivo</i> hinders the identification and analysis of this glycosylation. Here, we introduce TurboID to identify terminal sialic acid glycosylation in living cells. <i>Sp</i>CBM, the carbohydrate-binding domain of sialidase from <i>Streptococcus pneumoniae</i>, is fused with TurboID and overexpressed in HeLa cells. After streptavidin-based purification and detection by mass spectrometry, 31 terminal sialic acid N-glycosylated sites and 1359 putative terminal sialic acid glycosylated proteins are identified, many of which are located in the cytoplasm and nucleus.Efficient TurboID-based proximity labelling method for identifying terminal sialic acid glycosylation in living cells.Liu Wu W, Long Yunfeng Y, Bao Yongfen Y, Li Yang Y, Deng Mengxue M, Yang Xiuyi X, Zhu He H, Su Yanting YfalseEfficient TurboID-based proximate labeling method for identifying terminal sialic acid glycosylation in living cellsTurboID, a mutant biotin ligase, based proximity labeling method is a new efficient technique for recognizing protein-protein interactions and has been successfully applied to living cells. Sialic acids are typically the terminal monosaccharides attached to many glycoproteins and play many important roles in many biological processes. However, the lack of enrichment methods for terminal sialic acid glycosylation in vivo hinders the identification and analysis of this glycosylation. Here, we introduced this technique to identify terminal sialic acid glycosylation in living cells. SpCBM, the carbohydrate-binding domain of sialidase from Streptococcus pneumoniae, was fused with TurboID and overexpressed in HeLa cells. After streptavidin-based purification and detection by mass spectrometry (MS), we identified 41 terminal sialic acid N-glycosylated sites and 3065 putative terminal sialic acid glycosylated proteins which many of them were located in cytoplasm and nucleus.Thu Nov 04 00:00:00 GMT 2021PXD029556960636789692