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Stefanie Hauck, Christine von Toerne, Marcel Blindert, Jennifer Behler, Cornelia Deeg, Marius Ueffing; Quantitative Profiling of RMG Cell Surface Proteome Changes in Response to LPS Treatment. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5597.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal Müller glial cells (RMG) are active players during retinal diseases including inflammatory conditions such as uveitis and age-related macular degeneration. In order to gain fundamental understanding on RMG (re)actions during inflammation, we developed a proteomics approach screening specifically for cell surface and membrane protein reactions induced by prototype inflammatory inducer lipopolysaccharide LPS.
Sugar residues of cell surface proteins on intact MIO-M1 cells untreated or treated with LPS were mildly oxidized and covalently labelled with biotin. Biotinylated proteins were affinity purified and glycosylated peptides were specifically released by PNGase F. Label-free LC-MSMS was used for protein identification and quantification. Cell surface localization of identified proteins was validated by bioinformatic approaches and immunocytochemistry, LPS -induced changes were validated by western blots.
The cell surface biotinylation approach combined with quantitative and sensitive liquid chromatography mass spectrometry resulted in the identification of more than 500 proteins on MIO-M1 cell surface. Bioinformatic analysis allocates 75% of these proteins to be truly membrane or extracellular matrix proteins. This comprehensive cell surfaceome includes transmembrane receptors, transporters, adhesion molecules, signalling molecules and proteases, including 18 CD markers, 18 integrins, 41 solute carriers, two ephrins, five ephrin receptors and six plexins. Treatment of cells with LPS results in a highly reproducible significant shift of cell surface proteome with upregulation of 36 proteins and downregulation of 13 proteins. Among those LPS-induced cell surface expression changes are proteins that suggest an active role of these glial cells in inflammatory processes.
Cell surface biotinylation on glycosyl-residues in combination with label-free LC-MSMS is a sensitive and reproducible method to profile cell surface proteomics and sheds light on the biological properties of cells.
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