Identification of novel high mannose N-glycan isomers undescribed by current multicellular eukaryotic biosynthetic pathways

N-linked glycosylation is one of the most important post-translational modifications of proteins. Current knowledge of multicellular eukaryote N-glycan biosynthesis suggests high mannose N-glycans are generated in the endoplasmic reticulum and Golgi apparatus through conserved biosynthetic pathways. As a part of post-translational modifications, lipid dolichol-phosphate linked oligosaccharide Glc3Man9GlcNAc2 is transferred to proteins, and glucoses and mannose are sequentially removed by various ER- and Golgi-localized glucosidases and -1,2-mannosidases. According to reported biosynthetic pathways, four Man7GlcNAc2 isomers, three Man6GlcNAc2 isomers, and one Man5GlcNAc2 isomer are generated during this process. In this study, we applied our latest mass spectrometry method, logically derived sequence tandem mass spectrometry (LODES/MSn) to re-examine high mannose N-glycans extracted from various multicellular eukaryotes. LODES/MSn identified many high mannose N-glycan isomers previously unreported in plantae, animalia, cancer cells, and fungi. Importantly, their synthesis is yet described by known biosynthetic pathways, thereby suggesting additional and unidentified pathways for these N-glycans isomers in multicellular eukaryotic cells.