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Kyle A. Floyd, Landon S. Wilson, David M. Anderson, Kevin L. Schey, Om P. Srivastava, Stephen Barnes; Age-dependent Production of C-terminally Truncated αA-crystallin Species in Cataractous ICR/f and Non-cataractous Wistar Rats. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1304.
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Our previous mass spectrometry studies have shown the presence and spatial distribution of C-terminally truncated αA-crystallin species (at residues 151, 156, 157, 163, and 168) in the lens of the cataractous ICR/f rat. The goal of this study was to quantify (relatively) the amounts of the C-terminal truncations, compared to the intact full-length species, through different stages in development for both cataractous ICR/f and non-cataractous Wistar rats.
Lenses from ICR/f rats at 21, 50, and 100 days of age, and from Wistar rats at 21 and 100 days of age, were homogenized in 50mM ammonium bicarbonate/8M urea buffer. In-solution chymotryptic digestion was performed on 20μg of protein from each sample. Processed samples were resuspended in 0.1% formic acid with 1fmol of digested bovine serum albumin (loading control) and analyzed using an nanoLC-multiple reaction monitoring-MS method on an AB Sciex 5600 TripleTOF, for relative quantitation of the intact C-terminal peptide and C-terminal truncated species of the αA-crystallin.
αA-crystallin has a chymotryptic cleavage site at residue 141, yielding a 32 amino acid peptide for the intact C-terminus (residues 141-173). Resulting sub-peptides between residues 141-173 are products of C-truncation. Intensities of the full-length peptide were significantly higher (~2x or more) than all truncated species, with no substantial difference in amounts with age, or between animal models. C-truncations were slightly higher in the ICR/f rat than the Wistar, yet both showed the same patterns in age development. Intensities of C-truncations at residues 151, 163, and 168 were most abundant, and increased significantly with age in ICR/f (~4-5x from 21 to 100 days) and Wistar (~3-5x from 21 to 100 days) rats. Levels of the 156 and 157 truncations were lowest, with little change between 21 and 50 days in the ICR/f rat, but a drastic increase by day 100 (~4-5x). Levels of these truncations also increased in the Wistar rat from day 21 to 100 (~2-3x).
We have successfully developed a method to relatively quantify the full-length C-terminal peptide, and those of C-terminal truncations at residues 151, 156, 157, 163, and 168 from the αA-crystallin within the rat. These truncations increase with age from 21 to 100 days old in both the cataractous ICR/f and non-cataractous Wistar rats, while the amount of the full-length C-terminal peptide remains relatively constant with aging.
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