Analytical dissection of minor glycoforms and glycoprotein associations in rAAV preparations by multimodal glycoproteomics.

Accurate glycan analysis of viral vectors is essential for evaluating pharmaceutical quality. Recent advances in mass spectrometry-based analytical technologies have achieved glycosylation detection in adeno-associated viruses (AAVs). However, because only a minor subpopulation (< 1%) of recombinant AAV (rAAV) particles may carry glycans or associate with glycoproteins, distinguishing genuine AAV glycosylation from that of co-purified glycoproteins remains technically challenging, highlighting the need for analytical strategies that minimize glycan misassignment and reliably identify glycoprotein interactions. Here, we present a multimodal glycoproteomic approach to discriminate rare glycosylation events on rAAV capsids from glycosylated host-derived proteins associated with the particles. We employed an ultrasensitive lectin microarray coupled with a broadly reactive anti-AAV antibody to detect O-glycan-binding lectin signals in several rAAV preparations. Notably, a distinct signal was observed for Urtica dioica agglutinin (UDA). Subsequent liquid chromatography-tandem mass spectrometry, combined with UDA-based dual enrichment at both protein and peptide levels, identified a divalently high-mannose N-glycosylated peptide derived from the host AAV receptor (AAVR). Monovalent high-mannose N-glycopeptides of AAVR and Mac-2 binding protein were additionally detected using single-step protein-level enrichment, indicating an avidity-driven UDA binding mechanism. However, no N-glycosylation was detected on the rAAV capsids themselves. These findings underscore the value of integrated multimodal glycoproteomic workflows for resolving low-abundance glycosylated species and offer new insights into host-derived hitchhiker glycoproteins that may affect rAAV characterization and quality control.