JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST003746/files/AAV9_Empty_UDA_AspN_UDA1p2_Slot1-31_1_1323_uncalib_6.1.452_Byonic(N&O).xlsxhttps://storage.jpostdb.org/JPST003746/files/AAV5_Empty_UDA_AspN_UDA1p2_Slot1-30_1_1321_uncalib_6.1.452_Byonic(N&O).xlsxhttps://storage.jpostdb.org/JPST003746/files/6-8_UDA_AspN_UDA_1p2_Slot1-21_1_1213_uncalib_6.1.452_Byonic(N&O).xlsxhttps://storage.jpostdb.org/JPST003746/files/6-8_UDA_AspN_3p100_Slot1-19_1_1207_uncalib_6.1.452-(1)_Byonic(N&O).xlsxhttps://storage.jpostdb.org/JPST003746/files/AAV6_Empty_UDA_1p40_Slot1-23_1_1013(F006290).xlsxhttps://storage.jpostdb.org/JPST003746/files/AAV9_Empty_UDA_1p100_Slot1-14_1_1296_uncalib_6.1.452-(1)_Byonic(N&O).xlsxhttps://storage.jpostdb.org/JPST003746/files/AAV5_Empty_UDA_1p100_Slot1-13_1_1294_uncalib_6.1.452-(1)_Byonic(N&O).xlsxhttps://storage.jpostdb.org/JPST003746/files/AAV5_Empty_UDA_1p100_Slot1-13_1_1294(F006289).xlsxhttps://storage.jpostdb.org/JPST003746/files/AAV6_Empty_UDA_AspN_UDA9p10_Slot1-49_1_1054_uncalib_6.1.331_Byonic(N&O).xlsxhttps://storage.jpostdb.org/JPST003746/files/AAV6_Empty_UDA_1p40_Slot1-23_1_1013_uncalib_6.1.331-(4)_Byonic(N&O).xlsxhttps://storage.jpostdb.org/JPST003746/files/6-8_UDA_AspN_3p100_Slot1-19_1_1207(F006292).xlsxhttps://storage.jpostdb.org/JPST003746/files/AAV9_Empty_UDA_1p100_Slot1-14_1_1296(F006291).xlsxhttps://storage.jpostdb.org/JPST003746/files/6-8_UDA_AspN_UDA_1p2_Slot1-21_1_1213.d.ziphttps://storage.jpostdb.org/JPST003746/files/AAV9_Empty_UDA_AspN_UDA1p2_Slot1-31_1_1323.d.ziphttps://storage.jpostdb.org/JPST003746/files/AAV9_Empty_UDA_1p100_Slot1-14_1_1296.d.ziphttps://storage.jpostdb.org/JPST003746/files/6-8_UDA_AspN_3p100_Slot1-19_1_1207.d.ziphttps://storage.jpostdb.org/JPST003746/files/AAV5_Empty_UDA_1p100_Slot1-13_1_1294.d.ziphttps://storage.jpostdb.org/JPST003746/files/AAV6_Empty_UDA_AspN_UDA9p10_Slot1-49_1_1054.d.ziphttps://storage.jpostdb.org/JPST003746/files/AAV6_Empty_UDA_1p40_Slot1-23_1_1013.d.ziphttps://storage.jpostdb.org/JPST003746/files/AAV5_Empty_UDA_AspN_UDA1p2_Slot1-30_1_1321.d.zipprimaryOK200ProteomicsAtsushi KunoCloning Vector Aavhttps://repository.jpostdb.org/entry/JPST003746National Institute of Advanced Industrial Science and Technology (AIST)jPOSTfalseDeciphering Minor Glycan Signatures: Cross-Serotype Recombinant Adeno-Associated Virus (rAAV) Glycan Profiling Using Multimodal Glycoproteomics TechnologiesGlycan analysis of viral vectors is essential for pharmaceutical quality evaluation. Recent advances in mass spectrometry (MS)-based analytical technologies have identified glycosylation in adeno-associated viruses (AAVs). However, reported glycan structures vary between studies, due to the extremely low abundance (<1%) of glycosylated particles and the presence of hitchhiker molecules, making AAV glycosylation a topic of debate. This underscores the need for standardized and highly sensitive detection methods. Here, we present a multimodal glycoproteomic approach to characterize glycosylation across six serotypes of 28 recombinant AAVs (rAAVs). An ultrasensitive lectin microarray, using a broad-reactive anti-AAV antibody, detected O-glycan-binding lectin signals in rAAV6, consistent with previous findings. Notably, a unique binding signal was observed with Urtica dioica lectin (UDA). Liquid chromatography-tandem mass spectrometry (LC-MS/MS), combined with UDA-based glycoprotein/glycopeptide dual enrichment and glycopeptide identification, confirmed the predominant presence of a divalent high-mannose N-glycosylated peptide from a hitchhiking host-derived glycoprotein, the AAV receptor. In contrast, monovalent high-mannose N-glycosylated peptides were identified using a single-step protein-level enrichment approach, suggesting an avidity effect in UDA binding. These findings highlight the significance of advanced multimodal glycoproteomic in rAAV characterization and offer new insights into host-derived hitchhiker glycoproteins present in AAV preparations.Sat Apr 04 00:00:00 BST 2026PXD0625881116368