Small extracellular vesicles (sEVs) are lipid-bound particles ranging from 30 to 150 nm in size, released from cells to facilitate cell signaling processes. While glycosylation is a significant post-translational modification often linked with cell signaling, exploring the glycosylation of extracellular vesicle proteins remains challenging, with limited studies in this area.

In the present study, we developed a methodology to investigate the proteomic, N-glycopeptide, and chondroitin sulfate glycosaminoglycan profiles of sEVs derived from A549 lung adenocarcinoma and BEAS-2B non-tumorigenic cell lines. Before isolation, A549 cells were cultured in non-completed F12 medium for 72 hours, while BEAS-2B cells were cultured in BEGM medium for the same duration. The supernatant was then collected and subjected to centrifugation and filtering steps to remove cell debris, apoptotic bodies, and microvesicles. Small EVs were isolated by mini-size exclusion chromatography on custom-made columns, with fractions 3, 4, and 5 showing enrichment in sEVs and combined for further analysis.

Characterization of sEVs included transmission electron microscopy for shape assessment and microfluidic resistive pulse sensing for size distribution analysis. Isolated sEVs were subjected to repeated freeze-thaw cycles and were divided into two parts. One part underwent trypsin digestion followed by acetone precipitation to enrich glycopeptides in the pellet, while the supernatant contained non-glycosylated peptides. The other part was subjected to digestion with chondroitinase ABC enzyme to release chondroitin sulfate disaccharides. Following purification, the collected peptides, N-glycopeptides, and GAG disaccharides were analyzed by nanoUHPLC-MS(MS).

Six independent sEV isolations are currently performed from each cell line, with pure mediums serving as controls to exclude components not of cellular origin. Data analysis and subsequent statistical comparisons may reveal differences in proteomic, N-glycomic, and chondroitin sulfate profiles between the sEVs, suggesting potential clinical relevance of sEV glycosylation.

Support of the Lendület (Momentum) Program of the Hungarian Academy of Sciences and Semmelweis 250+ Excellence PhD Scholarship is acknowledged. The Authors are grateful to Markus Ralser for donating the nanoAcquity HPLC to the group.