Post-translational modifications of lens crystallins may decrease their stability and lead to protein denaturation, aggregation, light scatter, and cataract. Lens crystallins have been thoroughly analyzed by mass spectrometry to detect the major modifications. However, few studies have attempted to quantify the relative abundance of these modifications in normal and cataractous lenses, and determine if these modifications are associated with the light-scattering water-insoluble fraction. These measurements would allow testing the hypothesis that deamidation is the most important age-related modification in human lens, because it causes lens crystallins to unfold and expose normally buried cysteines that then undergo disulfide bonding to produce light scattering aggregates. The three aims below will test this hypothesis by: 1. determining the sites where deamidation and other modifications occur in lens crystallins so that their relative abundance can by measured within the water-soluble and water-insoluble proteins of both normal and cataractous aged human lens;2. determining the relative extent of oxidation at each specific cysteine residue in crystallins to determine if deamidation and disulfide bond formation are localized in the water-insoluble fraction and occur in regions that are normally buried, and 3. expressing mutant forms of crystallins containing deamidation sites occurring specifically in the water insoluble fraction of cataractous lenses, to determine if the deamidation increases the susceptibility of cysteines to disulfide bonding. The experiments will utilize two-dimensional chromatography and mass spectrometry to separate and analyze complex digests of crystallins from both normal and cataractous aged human lenses. Relative quantification of modifications will be determined by differential labeling of peptides with stable isotopes and the use of mass shifted internal standards. Informatics tools will be utilized so that the complex data analysis can be rapidly performed. Results may suggest strategies to slow the rate of cataract formation in humans by stabilizing deamidated crystallins, or preventing oxidation of their exposed cysteine residues.
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