Proteomic profiling of high purity extracellular vesicle subtypes from megakaryoblastic leukemia cells provides insights into myeloproliferative neoplasm biology

Vollständiger Abstract

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Aim: Extracellular vesicles (EVs) are emerging as important mediators in myeloproliferative neoplasms (MPNs), but the characteristics of distinct EV subtypes remain unclear. This study aimed to characterize the molecular heterogeneity of MEG-01-derived large EVs (lEVs) and small EVs (sEVs). Methods: lEVs and sEVs were isolated from MEG-01 cell culture medium by differential centrifugation followed by density gradient ultracentrifugation. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, immunoblotting, and on-bead flow cytometry. Quantitative proteomic analysis was performed to compare the protein cargo of lEVs and sEVs, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Results: Density gradient purification yielded high purity of lEV and sEV with distinct size, morphologies, and protein compositions. On-bead flow cytometry enabled efficient profiling of EV surface markers. Proteomic analysis revealed a shared core proteome between lEVs and sEVs, together with subtype specific protein signatures. Enrichment analyses indicated distinct molecular characteristics of the two EV populations, with sEVs showing enrichment of proteins associated with RNA-related processes and vesicle-mediated transport, whereas lEVs were enriched in proteins related to cytoskeletal organization and metabolism. Conclusion: MEG-01 derived lEVs and sEVs comprise molecularly distinct subpopulations with shared and subtype specific protein cargo. The workflow established in this study provides a robust platform for high purity EV isolation and characterization, and offers insights into MEG-01 EV heterogeneity relevant to megakaryocyte biology and MPN research.