Differentiation of peptide isomers and epimers by radical-directed dissociation

Isomerization and epimerization are spontaneously occurring modifications that represent common pathways for age-related damage in proteins. These modifications accumulate over time and are known to disrupt both structure and function, making their detection and identification an important goal in proteomic characterization. Unfortunately, because these modifications lead to no change in mass, they are typically overlooked in traditional proteomics. Herein we present protocols for mass-spectrometric identification and quantification of protein isomerization and epimerization. Illustrative examples are provided for analysis of crystallin proteins extracted from the eye lens. Crystallins provide an attractive model system for studying the age-related modifications of long-lived proteins due to the lack of protein turnover in the eye lens. We first outline procedures for tissue processing and protein extraction. We then describe methods for data acquisition and analysis for differentiation and identification of isomerized residues, including methods for quantifying isomers which cannot be chromatographically resolved.