Shi Yan Shi Yan Jorick Vanbeselaere Jorick Vanbeselaere Callum Ives Callum Ives David Stenitzer David Stenitzer Lena Nuschy Lena Nuschy Florian Woels Florian Woels Katharina Paschinger Katharina Paschinger Elisa Fadda Elisa Fadda Johannes Stadlmann Johannes Stadlmann Iain Wilson Iain Wilson American Society for Biochemistry and Molecular Biology 2025 Insect cells are a convenient cell factory to produce recombinant glycoproteins. Their glycosylation potential is believed to be simple, needing primarily addition of glycosyltransferases to humanize the recombinant products. In this study, the native glycoproteome of Spodoptera frugiperda Sf9 and Trichoplusia ni High Five cells, examined using an LC-MS/MS approach, revealed not only which proteins are N-glycosylated but also indicated that the N-glycomes contain novel glucuronylated and phosphorylcholine-modified glycans, in addition to typical oligomannosidic and fucosylated structures. These data were corroborated by a parallel MALDI-TOF MS/MS analysis of N-glycosidase-released oligosaccharides. Molecular modeling analysis of one endogenous Sf9 glycoprotein correlated the occurrence of complex and oligomannosidic N-glycans with the accessibility of the occupied N-glycosylation sites. Further, we showed that the N-glycans of influenza hemagglutinins and SARS-CoV-2 spike glycoprotein produced in Spodoptera cells possess a number of glycan structures modified with phosphorylcholine, but core difucosylation was minimal; in contrast, the Trichoplusia-produced hemagglutinin had only traces of the former type, while the latter was dominant. Detection of phosphorylcholine on these glycoproteins correlated with binding to human C-reactive protein. In conclusion, not just oligomannosidic or truncated paucimannosidic N-glycans, but structures with immunogenic features occur on both natural and recombinant glycoproteins derived from insect cell lines. PDFDocument eng 2026-01-29T10:46:54.783955Z 2025 Baculovirus Fucose Glucuronic Acid Insect Cells N-glycans Phosphorylcholine 5047475 b application/pdf http://creativecommons.org/licenses/by/4.0/