Evaluation of Extractants and Precipitants in Tear Film Proteomic Analyses

Abstract

To determine the efficiency of several protein extraction or precipitation treatments used in proteomic analyses. Tear samples were taken from each eye of 40 normal subjects using glass microcapillaries. Tear volume was measured followed by storage at -86°C. Lotrafilcon B contact lenses were fitted and worn for 14 days, followed by removal and storage at -86°C. Tear samples from each eye within a subject were randomly assigned to either one of four chemical treatments (acetone, trichloroacetic acid, urea, and trifluoroacetic acid/acetonitrile [TFA/ACN]) or no chemical treatment in groups of 10. Contact lens samples were subjected to the same treatments as tear samples for each subject, with a second treatment preceding the first. Protein concentrations were quantified by Bradford assay. For tear samples, a significant reduction in total protein was observed when subjected to any of the four treatments studied compared with those samples left untreated. A positive relationship was noted between protein concentration and tear volume for treated, untreated, and combined tear samples. For contact lens samples, there was a significant reduction in the amount of deposited protein removed when comparing acetone, trichloroacetic acid, and urea with TFA/ACN. A second extraction from contact lenses assigned to the urea and TFA/ACN groups yielded a significant amount of additional protein compared with the amount removed initially. Tear samples subjected to any of the evaluated chemical treatments provided significantly less protein than untreated samples. For contact lenses, TFA/ACN extraction provided the highest yield of available protein out of the four treatments evaluated.