Updated project metadata. Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved post-translational modification, whose biosynthesis is initiated by three non-redundant enzyme families, POMT1/POMT2, TMTC1-4 and TMEM260. In this study, we applied a targeted workflow for membrane glycoproteomics to five human cell lines and to a panel of genetically engineered cells with individual and combinatorial knock-out of O-Man glycosyltransferase genes to extensively map substrates, specificities, and crosstalk of O-Man enzymes. Our quantitative glycoproteomics results demonstrate new protein targets for the POMT1/POMT2 pathway and show that TMTC1-4 and TMEM260 widely target distinct Ig-like protein domains of plasma membrane proteins. This new evidence adds further knowledge on the emerging concept of domain-specific O-Man glycosylation and establishes a platform for functional studies of O-Man glycosylated adhesion molecules and receptors.