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Elod Kortvely, Eszter Emri, Sascha Dammeier, Marius Ueffing, Imre Lengyel; Cell layer specific proteome of the outer retina: laser capture microdissection of drusen and its cellular environment from paraffin embedded sections. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5247.
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© ARVO (1962-2015); The Authors (2016-present)
Drusen deposition is one of the early hallmarks of age-related macular degeneration (AMD), a leading cause of visual impairment and irreversible blindness. The molecular composition of drusen and its immediate surrounding tissues is not yet fully understood. In this study we analysed the proteome of paraffin embedded sectioned human eye tissues using laser capture microdissection to obtain cell layer specific information.
In this study we used paraffin embedded sections from 7 human post-mortem eyes (ages between 41 and 91) obtained from the pathology archive at UCL Institute of Ophthalmology. We isolated outer nuclear layer (ONL), photoreceptor outer segments (POS), retinal pigment epithelium (RPE), drusen and the Bruch’s membrane/choriocapillary complex (BrM) using laser capture microdissection (Zeiss PALM). Samples were catapulted into adhesive caps (Zeiss) and subjected to alternating cycles of ambient to high pressure using a Barocycler 2320EXT machine (Pressure BioSciences) in the presence of trypsin for ultra-fast protein digestion. Mass spectroscopic analysis was performed an Orbitrap Fusion (Thermo Scientific). Acquired spectra were analysed using Scaffold (Proteome Software).
We identified 206 proteins in ONL and 131 proteins in POS. Amongst these were Rhodopsin (RHO) and Retinal Outer Segment Membrane Protein 1 (ROM1) specific for photoreceptors. 412 proteins were identified in the RPE including the RPE specific Na+/H+ Exchange Regulatory Cofactor NHE-RF1 (SLC9A3R1) and Retinoid Isomerohydrolase (RPE65). The microdissected BrM complex, without drusen, contained 289 proteins with elastin (ELN) specifically identified only in this layer. In drusen 124 proteins were identified. Amongst these were vitronectin (VTN), clusterin (CLU) Tissue Inhibitor of Metallopeptidase 3 (TIMP3), Complement C9 (C9) and Apolipoprotein E (APOE).
The combination of laser capture microdissection and mass spectrometry is a powerful tool to detect cell and tissue specific protein profiles even from archived paraffin embedded and sectioned tissues. This approach has already enriched our knowledge on proteins present in the different layers. This approach, once used on tissue sections from diseased eyes, will contribute significantly to improve our understanding of the molecular changes underpinning the pathogenesis of AMD.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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