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Geeng-Fu Jang, Jack S Crabb, Bo Hu, Belinda Willard, Helen Kalirai, Arun D Singh, Sarah E Coupland, John W Crabb; Validation of Choroid from Uveal Melanoma Eyes as Control Tissue for Proteomic Studies. Invest. Ophthalmol. Vis. Sci. 2019;60(9):747.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the suitability of human choroid tissues harvested from enucleated eyes that contain uveal melanoma (UM) tumors to serve as control tissues for quantitative proteomic analyses of UM tumors.
Tryptic digests of choroid tissues from 13 eyes containing UM tumors were compared by quantitative LC MS/MS iTRAQ proteomic analyses with pooled tryptic digests of choroid tissues from 9 normal, disease-free eyes. Choroid specimens were excised at locations distant from the tumors and were from eyes containing 6 metastatic and 7 non-metastatic UM tumors. Tryptic digests (25 µg each) were labeled with unique iTRAQ tags, mixed, and fractionated by RPHPLC at pH10. Chromatography fractions were subjected to LC MS/MS on an Orbitrap Fusion Lumos Tribrid mass spectrometer. Protein identification and quantitation utilized the Mascot 2.6.2 search engine, the human UniProt database, the equal weight and weighted average analysis methods, and required a minimum of 2 unique peptides.
Total proteins quantified per specimen ranged from 1609-2348, with 1443 proteins present in all 13 specimens. Proteomic differences between choroid from eyes containing UM tumors and choroid from normal, disease-free eyes ranged from 9-11%. The mean ratio of the 1443 proteins in all 13 specimens was very close to unity (0.99) and less than 0.7% (10 proteins without adjustment for multiple comparisons) exhibited significant differences (p ≤ 0.05) between eyes containing Met and non-Met tumors.
The proteomic differences we detected between choroid from eyes containing UM tumors relative to choroid from disease-free eyes were very low and less than the variability that has been reported between a variety of normal tissues (2015 J Proteome Res 14, 2398; 2015 PLoS ONE 10: e0133270). The differences we observed between choroid from Met and non-Met donor eyes were also very low and significantly lower than those previously reported between Met and non-Met UM tumors (2015 PLoS ONE 10, e0135543). These results demonstrate that this set of choroid specimens from UM eyes is suitable to serve as a pooled reference control for determining proteomic differences between Met and non-Met UM tumors.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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