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A Saraf, JI Clark, JR Yates; Shortgun Identification of Post-translational Modification of Crystallins in Cataractous Lens by MudPIT Analysis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2386.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: : To map post-translational modifications of crystallins in a congenital cataract. Methods: Lens tissue obtained from a 4-year congenital cataract was homogenized and separated into soluble and insoluble fractions. The soluble fraction was denatured in 8M urea, pH adjusted to 8.5, reduced and alkylated. The reduced and alkylated proteins were divided into three equal fractions, and digested using three different enzymes; one of these enzymes cleaves in a site-specific manner while the other two cleave non-specifically. Due to the complexity of the sample each digest was analyzed separately using multidimensional chromatography and analyzed by a tandem mass spectrometer. The MS/MS spectra were interpreted and modifications identified using SEQUEST. Results: In the lens tissue a total of 240 proteins were identified with more than 40% sequence coverage. Post-translational modifications were mapped in crystallins and were found to contain a total of 74 modified sites. Modifications identified included Ser, Thr and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, Met, Tyr and Trp oxidations, amino acid sequence variations and proteolytic cleavages. Conclusion: These results show how MudPIT analysis can be used to provide information about different modifications from an extremely complex tissue like lens. This study should provide a basis for determining the modifications that are associated with cataractogenesis.
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