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L.L. David, A.E. Mixon, C. Smith, D.L. Rustvold; Quantification of Crystallin Composition in the Aged Human Lens by Methyl Esterification of Peptide Digests and Mass Spectrometry . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2137.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To develop a non-two-dimensional electrophoresis (2-DE) based methodology to quantify changes in the concentration of crystallins and their modifications during aging of human lens. Methods: The soluble and insoluble proteins from lenses of newborn, 1-month, 3-year, 18-year, 74-year, and 93-year old donors were denatured in 8M urea, reduced/alkylated, and digested with trypsin. Peptides from newborn lenses were then methyl esterified at all D and E residues, as well as the C-terminus using deuterated methanol (CD3OH) (d3). The remaining peptides from older lenses were esterified using normal methanol (d0). Equal amounts of the d3 esterified digests from newborn and each of the d0 esterified digests from older lenses were then mixed in equal proportions, separated by reverse phase chromatography, and alternating MS and MS/MS spectra acquired by electrospray ionization mass spectrometry. Peptides were then identified by automated analysis of MS/MS spectra using Sequest. The relative concentration of each crystallin peptide in the aged lens samples was then directly compared to the concentration found in newborn lenses by integrating the specific ion current of each mass shifted peptide during elution. For comparison, undigested protein from each lens sample was also separated by 2-DE. Results: Quantification of the changes in the relative abundance of crystallins by 2-DE during aging of human lens was impractical, because of numerous modified species of each crystallin that appeared with aging, and co-migration of several crystallin subunits within individual spots. Digestion of complex mixtures of lens proteins with trypsin, differential labeling with stable isotopes, and mass spectral analysis avoided these limitations. For example, the relative abundance of γS-crystallin, which was impossible to measure by 2-DE, was found to increase by a factor of 1.6 by 93 years of age in the soluble fraction compared to newborn lens, while the relative abundance of other crystallins decreased. The loss of ßB3 and ßA2 crystallins occurred first, since peptides from both were not detected past 3 years of age. Conclusions: Differential labeling of lens crystallin digests with stable isotopes will greatly simplify quantification of crystallins and their modifications during aging and cataract. The methodology can also be automated. This should allow the analysis of a greater number of lenses to determine which modifications are significantly associated with cataract.
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