A new method called SimpleCell technology has been developed to precisely map the human O-GalNAc glycoproteome. This method uses genetically engineered human cell lines to simplify O-glycosylation, allowing for the identification of O-glycan sites through mass spectrometry. The study analyzed 12 human cell lines from different organs and identified over 600 O-glycoproteins with nearly 3000 glycosites. An improved model, NetOGlyc4.0, was developed for predicting O-glycosylation. The results show that the O-glycoproteome is differentially regulated and dynamic, with unique subsets of O-glycoproteins in each cell line. The findings suggest that site-specific O-glycosylation plays a crucial role in protein function. The study also highlights the importance of understanding O-glycosylation in disease and its potential for discovering new biological functions. The SimpleCell strategy allows for the identification of O-glycosites in a wide range of proteins, including those with mucin-like domains and those with isolated sites. The study also identified O-glycosylation in Tyr residues, which may have special functions. The results provide a first-generation view of the human O-galNAc glycoproteome and demonstrate the potential for further research into the role of O-glycosylation in protein function and disease. The study also highlights the need for further research into the functions of individual GalNAc-T isoforms and the potential for developing targeted O-glycoproteomic strategies. The results suggest that O-glycosylation is a complex and dynamic process that is regulated by multiple factors, including the expression of different GalNAc-T isoforms. The study provides a comprehensive view of the human O-glycoproteome and highlights the importance of understanding O-glycosylation in biological processes and disease. The findings have important implications for the development of new therapies and the understanding of protein function.A new method called SimpleCell technology has been developed to precisely map the human O-GalNAc glycoproteome. This method uses genetically engineered human cell lines to simplify O-glycosylation, allowing for the identification of O-glycan sites through mass spectrometry. The study analyzed 12 human cell lines from different organs and identified over 600 O-glycoproteins with nearly 3000 glycosites. An improved model, NetOGlyc4.0, was developed for predicting O-glycosylation. The results show that the O-glycoproteome is differentially regulated and dynamic, with unique subsets of O-glycoproteins in each cell line. The findings suggest that site-specific O-glycosylation plays a crucial role in protein function. The study also highlights the importance of understanding O-glycosylation in disease and its potential for discovering new biological functions. The SimpleCell strategy allows for the identification of O-glycosites in a wide range of proteins, including those with mucin-like domains and those with isolated sites. The study also identified O-glycosylation in Tyr residues, which may have special functions. The results provide a first-generation view of the human O-galNAc glycoproteome and demonstrate the potential for further research into the role of O-glycosylation in protein function and disease. The study also highlights the need for further research into the functions of individual GalNAc-T isoforms and the potential for developing targeted O-glycoproteomic strategies. The results suggest that O-glycosylation is a complex and dynamic process that is regulated by multiple factors, including the expression of different GalNAc-T isoforms. The study provides a comprehensive view of the human O-glycoproteome and highlights the importance of understanding O-glycosylation in biological processes and disease. The findings have important implications for the development of new therapies and the understanding of protein function.