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LL David, AL McCormack, MA Riviere, KJ Lampi; Rapid Detection of Crystallin Deamidations in Human Lens by Two-Dimensional Chromatography and Mass Spectrometric Analysis of Peptide Methyl Esters . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3553.
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Purpose: To simplify the detection of age-related deamidation in crystallins. Methods: Whole soluble proteins from normal lenses of 55-year-old donors were reduced and alkylated, digested with trypsin, and subjected to methyl esterification by treatment with methanolic HCl. The complex digest was then separated by two-dimensional chromatography using a combination of cation exchange\reverse phase chromatography and MS/MS spectra of peptides acquired using an electrospray ionization ion trap instrument. Peptides were then identified by SEQUEST analysis of MS/MS spectra by taking into account the increased mass of methyl esters at the C-terminus of each peptide, and all Asp and Glu residues. Sites of deamidation were then detected by differentially searching for Gln and Asn residues with both normal masses or masses undergoing an increase of 15 units. Results: Automated identification of peptides from complex digests of whole lens soluble protein identified tryptic fragments from 15-72% of the total sequence of each crystallin present in human lens. The technique for the first time demonstrated the presence of beta A2 in human lens, which due to its low concentration, was previously undetected by two-dimensional electrophoresis. Deamidations at Q6, Q90, and Q104 of alpha A; N146 of alpha B; N81, N157, Q235 of beta B1; Q12, N15, Q70, N115, Q162, Q182, N204 of beta B2; Q51, Q83 of gamma C, and Q70, N53, and Q120 of gamma S were detected. Conclusion: The method allowed rapid detection of crystallin deamidations that were otherwise difficult to detect due to the single mass unit increase normally accompanying deamidation, and the presence of two or more potential deamidation sites per peptide. The method is also amendable to esterification with both normal and deuterated methanol, which will allow direct measurement of ratios of deamidation at specific residues in age-matched normal and cataractous lenses. Such information is important because deamidation may result in a loss of native crystallin structure, increased protein aggregation, and cataract.
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