Distinct proteomic and glycosylation signatures differentiate A549 tumor and BEAS-2B non-tumor cell line derived small extracellular vesicles

Extracellular vesicles (EVs) are central to intercellular communication and have gained attention as rich sources of molecular information in cancer research, but their molecular composition remains incompletely characterized. Protein glycosylation is a frequent post-translational modification; however, most EV studies focus on proteomics, while mapping glycosylation changes of proteins are still underrepresented. To address this gap, we analyzed the proteomic, N-glycoproteomic, and chondroitin/dermatan sulfate (CS/DS) glycosaminoglycan (GAG) profiles of small EVs (sEVs) derived from A549 lung adenocarcinoma and BEAS-2B non-tumorigenic epithelial cells. Principal component analysis and hierarchical clustering revealed that all three profiles strongly reflect sEV origin. Comparative proteomic analysis showed enrichment of proteins associated with cell cycle regulation, deoxyribonucleic acid repair, metabolism, and protein synthesis in A549 sEVs, while immune-related processes were enriched in BEAS-2B sEVs. Five differentially expressed CS proteoglycans were identified, highlighting the value of complementary GAG-level analysis. N-glycoproteomics revealed a shift from oligomannose to complex N-glycans in A549 sEVs. Prominent glycoproteins with multiple glycosylation sites included versican, galectin-3-binding protein and laminins. CS/DS content increased 3.4-fold in A549 sEVs, while the ratio of the two monosulfated disaccharides changed 2-fold. These findings demonstrate the utility of N-glycoproteomics and GAG profiling for sensitively characterizing molecular differences between sEVs derived from different cell culture models, thereby providing a foundation for future EV biomarker studies.