This study presents a comprehensive map of the human O-glycoproteome using a genetic engineering approach called SimpleCell, which simplifies O-glycosylation in human cell lines. The authors developed this method to enable proteome-wide discovery of O-glycan sites through 'bottom-up' ETD-based mass spectrometric analysis. They applied this approach to 12 human cell lines from different organs and identified over 600 O-glycoproteins with almost 3000 glycosites. The findings provide evidence that the O-glycoproteome is differentially regulated and dynamic across cell lines. The study also improved the NetOGlyc4.0 model for predicting O-glycosylation and identified unique subsets of O-glycoproteins in each cell line. The results highlight the importance of site-specific O-glycosylation in regulating protein function and suggest that further research is needed to understand the biological functions of these modifications.This study presents a comprehensive map of the human O-glycoproteome using a genetic engineering approach called SimpleCell, which simplifies O-glycosylation in human cell lines. The authors developed this method to enable proteome-wide discovery of O-glycan sites through 'bottom-up' ETD-based mass spectrometric analysis. They applied this approach to 12 human cell lines from different organs and identified over 600 O-glycoproteins with almost 3000 glycosites. The findings provide evidence that the O-glycoproteome is differentially regulated and dynamic across cell lines. The study also improved the NetOGlyc4.0 model for predicting O-glycosylation and identified unique subsets of O-glycoproteins in each cell line. The results highlight the importance of site-specific O-glycosylation in regulating protein function and suggest that further research is needed to understand the biological functions of these modifications.