Updated project metadata. We employed the newly available IMPa O-glycoprotease from Pseudomonas aeruginosa for O-glycoproteomics analysis of cultured cells and tissues. The glycopeptides were extracted, purified, and conjugated to a solid support before an enzymatic cleavage by IMPa. O-glycopeptides were analyzed by EThcD, which allowed for a site-specific and global analysis of O-glycans, especially those sialylated and the Tn antigen. Through wild-type or SimpleCell HEK293 cells, we present two approaches, one for the analysis of total O-glycoproteome and the other for the Tn glycoproteome, and showed that IMPa and EThcD are necessary for confident localization of O-glycans on glycopeptides. We applied this method to study the O-glycoproteome of mouse brain, which revealed the high frequency of sialylated O-glycans and the Tn antigen on brain glycoproteins.