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精准、全面的蛋白质组位点特异性糖基化解析用于蛋白质糖基化功能挖掘

Protein glycosylation is a heterogeneous posttranslational modification that generates great proteomic diversity and gives rise to functional variances. Precise and comprehensive characterization of protein glycosylation at site-specific level and the proteome scale is critical for understanding the biological functions. LC-MS/MS is the dominant tool for large-scale, site-specific glycoproteomic study. Efficient and robust mass spectrometry-based methods for the comprehensive analysis of intact glycopeptides are coveted. Recently, we proposed a series of qualitative and quantitative strategies for site-specific glycosylation analysis. Based on our previously developed pGlyco2.0 (Nat Commun., 2017, 8, 438), which is a one-step tandem MS strategy for intact glycopeptide identification with optimized stepped-energy fragmentation and a dedicated search engine, we developed pGlyco3 (Nat Methods, 2021, 18, 1515) that can comprehensively analyze intact N- and O-glycopeptides, including glycopeptides with modified saccharide units. To expand the discovery of pGlyco3.0, we developed pGlycoNovo (unpublished), a glycan database-free algorithm, and achieved generic and largest identification of intact N-glycopeptides in seven diverse model organisms with 45,503 site-specific glycans on 7856 glycoproteins. Recently, we developed a generic software tool, pGlycoQuant (bioRxiv 2021.11.15.468561), for accurate and convenient quantitative intact glycopeptide analysis, supporting both primary and tandem mass spectrometry quantitation for multiple quantitative strategies. pGlycoQuant enables intact glycopeptide quantitation with very low missing value via a deep residual network and includes a quantitative bioinformatics analysis module. A pGlycoQuant-based site-specific N-glycoproteomic study conducted by us quantifies 6435 intact N-glycopeptides in three hepatocellular carcinoma cell lines with different metastatic potentials, and together with invitro molecular biology experiments illustrate core fucosylation on the site 979 of L1 cell adhesion molecular (L1CAM) as a potential regulator of HCC metastasis. We have demonstrated the above methods as powerful tools for site-specific glycosylation exploration in biological and clinical samples and expect further applications in glycoproteomic studies.

191 2022-08-24

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