Development of a selective and sensitive analytical method to detect isomerized aspartic acid residues in crystallin using a combination of derivatization and liquid chromatography mass spectrometry.

The isomerization of amino acids in peptides and proteins induces structural changes and aggregation. The isomerization rate of aspartic acid (Asp) is high and causes various serious diseases including Alzheimer's disease and cataract. Herein, a method for the comprehensive separation and sensitive detection of isomerized crystallin containing Asp (l-α-Asp, l-ß-Asp, d-α-Asp, and d-ß-Asp) was developed using chiral derivatization and reversed-phase UHPLC separation. Of three candidate derivatization reagents tested for the separation of peptides containing isomerized aspartic acid, 2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazine-2-yl) pyrrolidine-2-carboxylate (DMT-(R)-Pro-OSu) was the most suitable reagent for separating isomerized peptides and improved the sensitivity of mass spectrometry by 50-fold. This method was applied to analyze heat-denatured crystallin. Asp58 and Asp151 residues in αA-crystallin (AAC) exhibited the highest isomerization rate in heated crystallin. Furthermore, the analysis of α-crystallin extracted from bovine eye lens identified isomerized Asp residues (Asp24/35, Asp58, and Asp151 in AAC and Asp140 in αB-crystallin (ABC)). These results indicate that the newly developed method using chiral derivatization provides selective and sensitive analysis of isomerized Asp sites in α-crystallin protein. This novel method will allow for the identification and quantification of isomerized amino acids in crystallin proteins.