Differential gel electrophoresis of the tear proteome.

PURPOSE

This study evaluates the use of differential gel electrophoresis (DIGE) for tear analysis and applies this technique to establish the effect of extended contact lens wear on the tear proteome.

METHODS

Flush tears were collected from nine healthy non-contact lens wearers at baseline, during the first day, and after 1 month of contact lens wear. Participants wore AIR OPTIX on an extended wear (EW) schedule. Tears were collected at mid-day and upon waking and analyzed for concentrations of total protein using the bicinchoninic acid assay method. DIGE was then performed to detect biomarkers likely to be affected by contact lens wear. Technique variability, physiological variability, and the effect of EW were established. Proteins with significantly changed abundance during lens wear were cut and identified with mass spectrometry.

RESULTS

Both Cy3 and Cy5 identified the same proteins relative to Cy2 with the highest fold difference of 4.4. The proteome varied by 3% at the same time point, with a significant increase in protein spots found between mid-day (257) and upon awakening (298). With EW, of the 311 proteins spots identified upon awakening, 15 differed significantly. Of the five differences between baseline and the first night of EW, all were greater at baseline. Of the differences between baseline and 1 month, six were greater at 1 month and four were greater at baseline.

CONCLUSIONS

DIGE is a repeatable proteomic technique capable of determining differences in the tear proteome. The extent of the participants' experience with lens wear is an important factor to consider when analyzing the tears from lens wearers, as neophytes appear to differ from those with experience.