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Binoy Appukuttan, Phillip A Wilmarth, Yuzhen Pan, Larry L David, Justine Smith; Inflammatory and neovascular markers identified by deep shotgun proteomic profiling of human retinal and choroidal vascular endothelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):373.
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Endothelial cells of the retina (REC) and choroid (CEC) have distinct protein phenotypes that may influence tissue-specific diseases. Characterizing the proteomic profiles of REC and CEC may allow identification of disease-associated cell-specific factors.
Cultures of REC and CEC were prepared, using Dynal magnetic beads linked to an anti-CD31 antibody, from 5 human cadaver donors. Cell proteins extracts (1mg) were digested and peptides were fractionated on a polysulfoethyl A cation exchange column. Fractions were analysed by reverse-phase LC-MS using an Agilent 1100 series capillary LC system and LTQ linear ion trap. BioWorks generated DTA files and SEQUEST (no enzyme, 2.5 Da tolerance, C+57 static mod) was used to assign peptide sequences to MS2 spectra with human Sprot target/decoy database. The PAW pipeline was used to control peptide error rates, and count peptide identifications and total protein spectral counts (SpC). Differential protein expression analysis was performed using the edgeR package in R. Differential expression candidates were selected based on Benjamini-Hochberg FDR-corrected p-values: high significance (P<0.01); medium (0.01≤P<0.05); low (0.05≤P<0.1); no difference (P≥0.1). EdgeR candidates were verified using log-transformed ratios of the average SpC re-normalized by a sliding window Z-transformation. Gene ontology (GO) and GO Slim annotations were compiled.
PAW processing identified 1,112,384 peptides from 5,281,964 MS2 spectra, mapping to 3,200-4,000 proteins per sample. Overall 4,942 proteins were identified in all REC and CEC donors. 2,521 (53%) proteins were observed in all 10 samples. Of the 280 proteins showing highly significant differential expression between EC types, 176 were significantly more abundant in REC and 104 were significantly more abundant in CEC. GO for “angiogenesis”, “inflammation” and “hypoxia” identified ANGPT2, CTGF, ADAMTS-1, ICAM1 and VCAM1 as highly expressed proteins in REC and NRP1, UACA and CAPN2 as more abundant in CEC.
Analysis of ocular EC-specific molecular phenotypes may provide insights into the pathogenesis of disease. The impact of differentially expressed angiogenic or inflammatory proteins will be tested in disease-relevant functional assays. These proteins may be targets for specific therapies of retinal or choroidal vasculopathies.
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