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K. Green–Church, K.K. Nichols, R.L. Pitsch, B.M. Ham, R. Perez, J.J. Nichols; Comparison of Proteomic Methods for Tear Profiling . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3535.
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The aims of this paper are to demonstrate three different proteomic methods and determine a comprehensive list of proteins found in the tear from normal patients. The data acquired from this study will be used to probe differences in protein expression levels from dry eye patients.
Tear samples were pooled and subjected to three types of proteomic analyses: 1D–SDS–PAGE followed by standard protein identification of gel slices by LC–MS/MS, 2D–SDS–PAGE followed by standard protein identification of gel spots by LC–MS/MS and finally, shot–gun proteomics. As many bands/cores as could be detected from the 1D and 2D SDS–PAGE were cut out, digested with trypsin and analyzed by tandem mass spectrometry for identification by the generation of sequence tags and database searching. For shot–gun proteomics, tear samples were digested with trypsin directly with out gel separation and proteins were identified using tandem mass spectrometry and database searching.
Tear samples were initially analyzed by 1D–SDS–PAGE. From the 11 regions analyzed from the gel, a total of 30 distinct proteins were identified with several proteins observed in more than one band of the gel. This method produced the most protein identifications between the three methods and also revealed potential protein complexes. Tear samples were next examined by 2D–SDS–PAGE. Evaluation of the spot pattern revealed several protein modifications based on the multiple spots observed at similar molecular weight but different pI. For example, the multiple spots along the 80 KDa region are predicted to be glycosylated lactoferrin. De–glycosylation of the tear samples significantly condensed the spots revealing that many of the proteins in the tear are heavily glycosylated.
Finally, tear samples were analyzed by a shot–gun proteomic approach. Fourteen proteins were identified via this method. This method, which possesses the ability to determine relative quantities, showed the three most abundant proteins of the fourteen identified to be lactoferrin > lipocalin > lysozyme.
Three proteomic methods were utilized to examine the proteins found in the tear. All methods are effective examining protein profiles and each method provides a different insight regarding total protein identification, modification analysis or relative quantitation.
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