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E. A. Hoffman, K. L. Schey, W. D. Stamer; Protein Expression Pattern of Trabecular Meshwork Cell Exosomes. Invest. Ophthalmol. Vis. Sci. 2010;51(13):155.
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© ARVO (1962-2015); The Authors (2016-present)
To define the protein expression pattern of purified exosomes from conditioned media of human trabecular meshwork (HTM) cells.
Exosome samples were purified by differential centrifugation from conditioned media of mature HTM cell monolayers (two cell strains from two individual donor eyes) or from human urine (as control), solubilized in SDS sample buffer and run into an SDS-PAGE gel. Following in-gel tryptic digestion of excised gel regions, extracted peptides were subjected to two dimensional HPLC separation (strong cation exchange and reverse phase) and tandem mass spectrometry analysis (MS/MS) using the multidimensional protein identification technology (MudPIT) strategy. MS/MS spectra were searched against a human protein database using Sequest for protein identification. Protein expression was confirmed by Western blot analyses in exosomes from 5 different HTM cell strains.
HTM cell and urine exosomes contained many of the same proteins, including previously characterized exosome markers such as membrane organizers (annexins1, 2, 4-7, 11, flotillin and myoferlin), tetraspanins (CD9, CD81, syntenin-1) as well as integral membrane proteins and ESCRT proteins involved in vesicular trafficking. Interestingly, Myocilin was uniquely expressed by HTM cell exosomes.
While myocilin expression appears distinctive, exosomes from HTM cells contain proteins that characterize exosomes in other cell types.
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