Abstract
Purpose :
Age-related macular degeneration (AMD) is a leading cause of vision impairment among the elderly worldwide and there is evidence that EMT may be involved in AMD pathogenesis in vivo. The advent of embryonic stem (ES) cell-derived RPE cells enables us to investigate EMT-related pathways in human RPE cells in a tightly controlled and systems-based manner. The experiments outlined here seek to discern the key protein actors and phosphorylation events in RPE EMT to aid in development of novel therapeutic approaches to modulating this process.
Methods :
Human ES cells were cultured in mTeSR with fibroblast growth factors, followed by differentiation media containing 15% KO serum. Differentiated cells matured in RPE medium with B27 serum free supplement. RPE monolayers under five conditions were examined in duplicate: 1) untreated monolayer; 2) enzymatic dissociation and replating for 1hr; 3) enzymatic dissociation + 3hr replating; 4) treatment with TGFb and TNFa (TGNF) for 1hr; 5) TGNF treatment for 3hr. Protein was then harvested and prepared for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Phosphorylated peptides will be extracted with Fe3+ immobilized metal affinity chromatography for phosphoproteome analysis and flowthrough collected and fractionated for global proteome analysis. We will analyze phospho- and global proteome fractions on Orbitrap MS machines and perform peptide sequence matching and quantification at the CPD at JHMI.
Results :
Preliminary proteomics data from dissociation-induced RPE EMT quantified over 8000 proteins. These data show significant increases in pathways that regulate transcription and translation, and in kinases such as mitogen activated protein kinases (MAPKs) and aurora kinases. We expect phosphoproteome data to identify >20,000 phosphorylation sites and to be particularly informative to the key signaling events in RPE EMT.
Conclusions :
Systems-based analysis of ES-derived RPE cells during EMT may provide useful biological insights and help identify potential therapeutic targets. The multi-timepoints and dual means of EMT induction allow comparison these methods and narrow down key players in EMT with higher precision, helping limit erroneous associations between proteome perturbations and EMT and bolstering those exhibiting stronger association.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.