# Distinct Proteomic and Glycosylation Signatures Differentiate A549 Tumor and BEAS-2B Nontumor Cell Line–Derived Small Extracellular Vesicles

## TL;DR

The study compares protein and sugar patterns in small vesicles from cancerous and non-cancerous cells to reveal distinct molecular signatures.

## Contribution

The work introduces glycosylation profiling as a novel method to distinguish tumor-derived extracellular vesicles from non-tumor ones.

## Key findings

- A549 sEVs show enrichment in complex N-glycans and proteins related to cell cycle and metabolism.
- CS/DS content is 3.4-fold higher in A549 sEVs compared to BEAS-2B sEVs.
- Glycan-level analysis provides greater sensitivity than proteomics alone in differentiating sEV origins.

## Abstract

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, whereas mapping glycosylation changes of proteins is still under-represented. To address this gap, we analyzed the proteomic, N-glycoproteomic, and chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycan (GAG) profiles of small EVs (sEVs) derived from A549 lung adenocarcinoma and BEAS-2B nontumorigenic 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, DNA repair, metabolism, and protein synthesis in A549 sEVs, whereas 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, whereas the ratio of the two monosulfated disaccharides changed twofold. 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.

•Integrated proteomics, N-glycoproteomics, and CS/DS GAG profiling of sEVs.•A549 and BEAS-2B sEVs show distinct protein and glycosylation signatures.•A549 sEVs are enriched in complex type N-glycans.•CS/DS abundance and sulfation patterns differ markedly between the two cell lines.•Glycan-level analyses add sensitivity beyond conventional EV proteomics.

Integrated proteomics, N-glycoproteomics, and CS/DS GAG profiling of sEVs.

A549 and BEAS-2B sEVs show distinct protein and glycosylation signatures.

A549 sEVs are enriched in complex type N-glycans.

CS/DS abundance and sulfation patterns differ markedly between the two cell lines.

Glycan-level analyses add sensitivity beyond conventional EV proteomics.

Small extracellular vesicles (sEVs) carry molecular information reflective of their cells of origin, but the glycosylation of sEV proteins remains underexplored. Here, we combine proteomics, N-glycoproteomics, and chondroitin sulfate/dermatan sulfate analysis to compare sEVs from lung adenocarcinoma (A549) and nontumorigenic epithelial (BEAS-2B) cells. Distinct glycopeptide and chondroitin sulfate/dermatan sulfate profiles clearly differentiate between A549 and BEAS-2B sEVs, demonstrating the added value of glycoanalytical workflows for EV characterization and biomarker discovery.