Tissue Context Shapes N-Glycan Processing and Site-Specific Microheterogeneity in Adult Drosophila melanogaster Compared With S2 Cells.

N-glycosylation is an important regulator of insect development, tissue organization, neural function, and other physiological processes, yet the extent to which cultured insect cells reproduce organismal glycosylation remains poorly understood. Here, we characterized the whole-body N-glycoproteome of adult Drosophila melanogaster and compared it with our previously generated site-specific glycoproteome of Drosophila S2 cells. Intact glycopeptide analysis by LC-MS/MS identified 2144 glycopeptides representing 526 glycoproteins, 875 N-glycosylation sites, and 38 glycan compositions in adult flies. Both adult flies and S2 cells retained a broadly conserved glycan repertoire dominated by oligomannose-type structures. However, this global similarity concealed substantial differences at the protein, site, and glycan levels. Only 34.6% of glycoproteins and 32.7% of glycosylation sites were shared between the two systems. Adult flies showed markedly higher representation of difucosylated glycans (12.0% vs. 0.006% in S2 cells), including glycan structures associated with neural glycoproteins, and exhibited lower overall site-specific microheterogeneity. At individual conserved sites, glycan processing could differ strikingly between systems, demonstrating that glycosylation is strongly influenced by organismal and cellular context. These findings establish a whole-body adult Drosophila N-glycoproteomic reference and show that S2 cells reproduce the core insect glycosylation program but only partially represent the protein- and site-specific organization of glycosylation in the intact insect. The dataset provides a resource for investigating glycan-dependent insect physiology and offers a useful benchmark for evaluating insect cell systems used in glycoprotein biotechnology.