Purchase this article with an account.
Viridiana Kocaba, Vidhya Venkatraman, Dawn Neo, Gary S L Peh, Jodhbir S Mehta; Comparison of the protein composition between fresh and frozen acellular Descemet’s membranes. Invest. Ophthalmol. Vis. Sci. 2021;62(8):821.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We compared the protein composition of completely decellularized Descemet’s membrane (DM), using 2 different sample preparation methods. The DMs derived from paired donor cornea were either subjected to freeze-thaw cycle or not, before mass spectrometry (MS) analysis.
Two corneas were subjected to a freeze-thaw cycle to remove corneal endothelial cells (CECs) by freezing the cornea at -20° C for 48 hours and thawing it gently at RT while the other two corneas from the fellow eye were incubated with culture media at 37°C for 48 hours. The corneas were washed with cell culture media to remove all the CECs. The DMs were peeled and the proteins were extracted with 2% SDS in 100 mM Tris buffer using a bullet blender homogenizer (Next Advance, NY). The supernatant was collected from homogenized sample. Extracted proteins were quantified by BCA method and subjected to SDS-PAGE fractionation. Protein bands were excised and subjected to in-gel trypsin digestion. The digested peptides were used for LC-MS/MS analysis performed using a Q Exactive mass spectrometer coupled with an online Dionex Ultimate 3000 RSLC nanoLC system (Thermo Scientific; MA). Tryptic peptides were separated with a Dionex EASY-spray column (PepMap® C18, 3µm, 100Å) using a typical 120min gradient of mobile phase A (0.1% FA) and mobile phase B (0.1% FA in ACN) with a flow rate of 300nl/min.
The LC-MS/MS-based analysis identified more proteins in the DMs that were subjected to the freeze-thaw cycle than the ones without the freeze-thaw cycle. A total number of 951 proteins were identified in the freshly processed group and a total of 1423 proteins were identified in the tissue subjected to freezing. The freeze-thaw cycle identified 33% more proteins compared to the samples that were processed fresh with mild enzymatic digestion. A total of 53 proteins were common among all samples in the freshly processed group while 117 proteins were common in the sample group subjected to free-thaw. The common proteins identified in all the samples were also analyzed manually using the Uniprot database for the sub-cellular localization and 98% of the protein were classified as extracellular or secreted proteins confirming that the identified proteins truly belong to the acellular DM.
The DMs that underwent a freeze-thaw cycle resulted in more proteins than DMs that were prepared fresh and subjected to mild enzyme digestion.
This is a 2021 ARVO Annual Meeting abstract.
This PDF is available to Subscribers Only