Abstract
Abstract: :
Purpose: To globally characterize the extent of disulfide bond formation of specific cysteine residues in water–soluble (WS) and water–insoluble (WI) crystallins from a 93–year old nuclear cataract. Methods: A single lens from a 93–year old male with nuclear cataract was homogenized and the proteins separated into WS and WI fractions. An age–matched normal lens was used as a control. Each sample was divided into two equal portions. One portion was labeled with the light form of the isotope coded affinity tag (ICAT) reagent without reduction, and the other portion first reduced and then labeled with the heavy ICAT reagent. The labeled proteins were recombined, trypsin digested, cysteine containing peptides affinity purified, and analyzed by reverse–phase LC–MS/MS in triplicate. Peptides were identified with SEQUEST software and isotopically–labeled peptide pairs were analyzed using quantitative software developed in–house. The relative abundance of individual crystallins in the WS and WI fractions of the cataractous lens was estimated from two–dimensional chromatography/mass spectrometry by summing the number of MS/MS spectra assigned to each protein. Results: Thirty–four cysteine residues were observed. Quantitative data on 20 peptides containing 25 cysteine residues was obtained. The overall average extent of disulfide bonding increased going from normal WS, normal WI, and cataract WI by 24, 44, and 67%, respectively. The susceptibility of specific cysteines to disulfide bonding in the WI fraction of cataractous lenses ranged from 30% at residues 131/142 in αA and residue 37 in ßB2, to 94% at residue 153 in γC. Estimation of the relative abundance of crystallins in the WS and WI fractions of cataractous lenses also indicted that γ–crystallins, the most disulfide–bonded crystallins, were also selectively insolubilized. In contrast, ßA3/A1, ßA4, and ßB1, with intermediate degrees of disulfide bonding, were abundant in both fractions, and ßB2, the lease disulfide bonded ß–crystallin, was selectively found in the WS fraction. Conclusions: The association of disulfide bonding with crystallin insolubilization suggested that it may either directly contribute to insolubilization, or that cysteine residues in the WI crystallins are more exposed and have greater susceptible to oxidation. Since there was little correlation between predicted solvent accessibility of cysteine residues in γ–crystallins and the extent of oxidation, disulfide bonds may form after denaturation has occurred.
Keywords: cataract • crystallins • proteomics