PURPOSE: To determine the efficiency of several protein extraction or precipitation treatments used in proteomic analyses. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
RCT Entities:
PURPOSE: To determine the efficiency of several protein extraction or precipitation treatments used in proteomic analyses. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.