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K. B. Green-Church, L. Zhang, K. K. Nichols; Comparison of Protein Extraction Techniques From Tears Collected by Schirmer Strips for Proteomic Analysis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4315.
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We report the differences in protein identification using eight different extraction techniques from tears collected by Schirmer Strips. The effect of separating the bulb from the Schirmer Strip verses using the entire strip was examined.
Human tear samples from non-dry eye patients were collected from Schirmer strips and stored at -80oC. The extraction buffers were Buffer A: 0.9% w/v NaCl in phosphate Buffer; Buffer B: 0.9% w/v NaCl in phosphate Buffer with 0.25% NP40 and 0.25% ABS-14; Buffer C: 100mM ABC; Buffer D: 100mM ABC with 0.25% NP40 and 0.25% ABS-14; Buffer E: 40mM Tris-HCl, 7M Urea, 2M Thiourea, 0.25% NP40 and 0.25% ABS-14. The use of mass spectrometry compatible surfactants Invitrosol, Rapigest and ProteaseMax were also investigated and the bulb portion of the Schirmer strip was cut away. Extraction was performed for one hour and the proteins were precipitated with acetone. The total protein recovery was measured by Bradford Assay and the remaining protein sample was digested with trypsin. Capillary-liquid chromatography-nanospray tandem mass spectrometry (Capillary-LC/MS/MS) of digested protein mixture was performed on a Thermo Finnigan LTQ orbitrap mass spectrometer equipped with a microspray source operated in positive ion mode.
The extraction conditions were found to have a noteworthy effect on the protein amounts extracted and subsequent protein identification. The total protein amounts recovered ranged from 10.7 µg - 36.3 µg and the number of proteins identified ranged from 150 proteins to 314. Table 1 summarizes the results of all the extraction methods examined. The proteins identified were sorted according to function and compared across extraction methods.
Notable differences in protein recovery and subsequent protein identifications were observed. 100mM ABC with proteaseMAX identified the most proteins. Extracting proteins from the bulb alone gives identifies more proteins than the strip alone. These results are critical for future proteomic studies of tears collected by Schirmer strips from dry eye patients compared to normal patients.
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