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S. M. Hauck, M. Irmler, S. Schoeffmann, P. del Rio, H. Sarioglu, J. Beckers, M. Ueffing; Secreted Proteins From Primary Retinal Mueller Glial Cells: A Combined Genomics and Proteomics Approach. Invest. Ophthalmol. Vis. Sci. 2009;50(13):670.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal Mueller glial cells (RMG) are recognized as intrinsic source of neuroprotective factors supporting retinal neurons. We aim at identifying as yet unknown secreted factors from RMG in order to develop future therapeutic strategies against neurodegeneration.
Primary RMG are isolated from young adult pig retinas by papain digestion and subsequently purified by panning. Total RNA is isolated from RMG directly after panning or after extended time in culture (two weeks = day14 or three weeks = day21) and used for Affymetrix GeneChips analysis on porcine arrays. Additionally, for direct analysis of secreted proteins in the extracellular compartments, conditioned media from RMG day14 and day21 are collected, labelled with isotope-coded protein labels (ICPL) and tryptically digested. Combined samples are subject to LC-MSMS mass spectrometric analyses (OrbiTrap), proteins are identified, validated by stringent scaffold analyses and quantified through the differential mass tags.
Based on porcine Affymetrix GeneChips we found RMG expressing (average expression ≥100) 8529 probesets on day 1, 8549 probesets on day 14 and 8525 probesets on day 21. Among these, a total of 216 non-redundant transcripts coded for secreted proteins in RMG day1, 241 in RMG day14 and 233 in RMG day21. Quantitative mass spectrometric measurements from extracellular compartments of RMG day 14 and day 21 resulted in the identification of more than 250 proteins. Of these, roughly 80 proteins contain a secretion signal as predicted by SignalP 3.0. Relative expression based on array results as well as quantification of proteins by ICPL labelling of the secreted proteome enables to predict the expression profile along with culture time for primary RMG. Whereas the expression profile changes dramatically from d1 to d14, afterwards expression levels are mostly stable.
The combined approach of transcriptome and targeted proteome analysis enables the identification of a large set of secreted proteins from primary RMG. Among these are well-known extracellular components, like SPARC, osteopontin, MMPs and FGFs, as well as many novel secreted factors, some of which are currently being evaluated towards neuroprotective activity.
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