June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Comparison of the protein composition between fresh and frozen acellular Descemet’s membranes
Author Affiliations & Notes
  • Viridiana Kocaba
    Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore, Singapore
    NIIOS, Netherlands Institute for Innovative Ocular Surgery, Rotterdam, Netherlands
  • Vidhya Venkatraman
    Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore, Singapore
  • Dawn Neo
    Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore, Singapore
  • Gary S L Peh
    Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore, Singapore
  • Jodhbir S Mehta
    Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore, Singapore
    Singapore National Eye Centre, Singapore, Singapore, Singapore
  • Footnotes
    Commercial Relationships   Viridiana Kocaba, None; Vidhya Venkatraman, None; Dawn Neo, None; Gary Peh, None; Jodhbir Mehta, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 821. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      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)

      ×
  • Supplements
Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×