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X. He, J. Jacob; Dry versus Normal Rabbit Tear Proteomics Using Micro-HPLC Techniques. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5296. doi: https://doi.org/.
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To compare the differences in rabbit tear protein profiles collected using Schirmer strips and capillary tubes and identify key changes in the protein profile of dry eye rabbit tears compared with normal eye tears using 1D and 2D HPLC.
Rabbit tears were collected by standard Schirmer strip methodology and silanized micro-capillary tubes. Samples and strips were stored individually at -80°C in eppendorf tubes until use. Total protein content (TPC) was determined for each collection type. Tears from the Schirmer strips were successively rinsed and lightly centrifuged using 20 mM phosphate buffer (pH 7.4) 4 times. The filtrates were pooled. Another centrifugation was applied to remove any cells. The TPC of the resulting supernatant and the capillary-collected tears was determined using a standard micro-BCA protein assay protocol.Protein profiling by HPLC: for 1D HPLC, H2O/ACN solvent with 0.05% TFA was applied as mobile phase using a C18 reverse column; for 2D HPLC, a strong cation exchange column and high salting buffer were used followed by a reverse C18 column.
The relationship between tear uptake volume and the indicating scale (mm) on the Schirmer strips was calculated. Therefore, the protein concentration for Schirmer strip-taken tears could be evaluated. Cell adhesion on Schirmer strips was observed through cell staining. This staining confirmed the presence of cellular proteins in tears. There was no striking difference in total protein content between dry eye tears and contralateral eye tears. However, qualitative comparison by HPLC of the protein profiles of dry eye tears showed a remarkable difference in the proportion of various protein peaks as compared with the profiles of control eye tears. Approximately 40 peaks of tear protein were obtained. Several abundant proteins were assigned from protein standards.
The TPC in Schirmer strip-taken rabbit eye tears was successfully calculated. The protein profiles from novel micro-HPLC revealed differences between dry eye tears and normal eye tears. Micro-HPLC makes feasible the characterization of dry eye-related tear proteins from individual tear samples and therefore facilitates tear proteomic research.
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