Extracellular vesicles such as exosomes are generally covered with an array of glycans, which are controlled by the host-cell glyco-synthetic machinery, similar to secreted and membrane glycoproteins. Several exosome subpopulations classified by their tetraspanin expression have been investigated in the context of diseases. However, a comparative analysis of their glycomics has never been attempted. Herein, we report a method for the comparative glycomic analysis of exosome subpopulations among pancreatic cancer cell lines. Glycomic profiles were obtained for extracellular vesicles, secreted glycoproteins, and membrane glycoproteins from eight cell lines. Statistical analyses revealed high populations of PHA-L-binding proteins in the vesicles. The surfaces of extracellular vesicles were labeled with Cy3 and captured by magnetic beads with antibodies against tetraspanins (CD9, CD63, and CD81). The coprecipitated vesicles were lysed and subjected to a lectin microarray analysis. A hierarchical clustering analysis using 19 glycomic profiles confirmed that most subpopulations, except CD81-positive exosomes, could be distinguished according to the host-cell species. Principal component analysis and subsequent lectin-affinity capturing of intact exosomes highlighted that CD81-positive exosomes preferentially expressed not PHA-L- but LEL-binding proteins on their surfaces. These data suggested that exosomal glycomics depended on the host-cell type and subpopulation.
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