Identification of Isomeric Aspartate residues in βB2-crystallin from Aged Human Lens

Many post-translational modifications such as oxidation, deamidation and isomerization of amino acid residues occur in lens proteins with aging. One such modification, isomerization of aspartate in lens alpha-crystallin, has been well studied by amino acid enantiomer analysis and LC-MS/MS. LC-MS/MS can quickly and easily identify D- and L-amino acid-containing peptides without purification of lens protein mixtures. However, this method has a weak point in that isomeric peptides of major components are detected predominantly, while those from minor proteins such as beta- and gamma-crystallins have not been fully determined. Therefore, the isomerization of amino acid residues in beta- and gamma-crystallin families has been little studied. To solve those problems and detect the isomerization of Asp residues in lens beta B2-crystallin, the main component of the beta-crystallin family, here we have developed steps for sample fractionation before D/L analysis based on either LC-MS/MS or amino acid derivatization to diastereoisomers followed by RP-HPLC. To capture a small amount of peptide, a multiple reaction monitoring (MRM) method based on quadrupole MS/MS (Q-MS) was applied to the water-soluble fraction of whole lens. The D/L analysis based on both LC-MS/MS and diastereoisomer formation showed the presence of multiple isomerization sites, including Asp4, Asp83, Asp92 and Asp192, in beta B2-crystallin in aged lens. These isomerization sites were confirmed to exist in an age-dependent manner by Q-MS. Synthetic peptides of beta B2-crystallin containing different isomers of Asp showed differential elution profiles during RP-HPLC, indicating differences in the local structure or hydrophobicity of Asp-isomer-containing peptides. These results suggest that the isomerization sites are distributed on exposed regions of beta B2-crystallin and thus likely to have an impact on crystallin subunit subunit interactions, induce abnormal crystallin aggregation, and contribute to senile cataract formation in aged lens.