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Pierre Roy, Javier Soria, Jaime Etxebarria, Tatiana Maria Suarez-Cortes; Optimization of a protein extraction method compatible with proteomic approaches using new device for conjunctival impression. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2763.
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© ARVO (1962-2015); The Authors (2016-present)
Conjunctival epithelium plays a key role in determining ocular surface condition. However, there is a lack of investigation of the role of conjunctival proteins in ocular surface pathophysiology, mainly due to the scarce cellular material obtained by conventional impression cytology (CIC). The novel conjunctival epithelium collection tool EYEPRIM (OPIA Technologies) may offer new perspective. In this study, protein recovery using EYEPRIM and CIC techniques was compared. Several extraction buffers were evaluated to verify their compatibility with different proteomic approaches.
A total of 32 conjunctival samples were collected using EYEPRIM and 29 using CIC. Samples were collected from healthy volunteers at the Cruces Hospital (Spain). Protein extraction from CIC was performed using urea based buffer (UBB). EYEPRIM samples were subjected to protein extraction using 4 different buffers: i) UBB, compatible with proteomic analysis; ii) three immunoassay compatible buffers (ICB1, ICB2, and ICB3). Protein recovery yield comparison between sample collection method and extraction strategies was carried out. Bidimensional electrophoresis with proteins extracted using UBB and immunodetection assays of ACTIN, ELAM and CDH1 proteins by dot-blot were performed to validate compatibility with proteomic techniques.
Protein recovery was improved with EYEPRIM compared to CIC using UBB buffer: 77.14 ± 27.18μg and 13.96 ± 8.11μg respectively (p-value < 0.0001). EYEPRIM with UBB obtained the best total protein recovery (77.14μg) in comparison with the ICB buffers assayed (43.5μg, 47.72μg and 48.73μg for ICB1, ICB2, ICB3 respectively). No significant differences in protein recovery were found between the ICBs employed. The 2-DE gel resolved 1263 spots using UBB and EYEPRIM. Dot-blot analyses showed compatibility with ICBs and higher detection signal using ICB2, which indicates superior antigen recognition.
EYEPRIM provides better protein recovery respect to CIC, independently of the buffer. Conjunctival sample collection by EYEPRIM using UBB for protein extraction offers an optimal strategy for proteomic analysis. ICB buffers makes extraction procedure compatible with immunoassays even if lower protein recovery is achieved. Depending on the goal of each study, it should be determined the most suitable protein extraction method.
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