Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation

Relative quantitation of aspartyl and isoaspartyl residue mixtures from asparagine deamidation is demonstrated using electron capture dissociation without prior HPLC separation. The method utilizes the linear relationship found between the relative abundance of the isoaspartyl diagnostic ion, z(n)-57, and % isoaspartyl content based on the ECD spectra of known isoaspartyl/aspartyl mixtures of synthetic peptides. The observed linearity appears to be sequence independent because the relationship exists despite sequence variations and changes in backbone fragment abundances when isoaspartyl and aspartyl residues are interchanged. Furthermore, a new method to calculate the relative abundances of isomer from protein deamidation without synthetic peptides is proposed and tested using a linear peptide released by protein digestion that contains the deamidation site. The proteolytic peptide can be rapidly aged to the expected 3:1 (isoaspartyl:aspartyl) mixture to generate a two-point calibration standard for ECD analysis. The procedure can then be used to determine the relative abundance of deamidation products from in vivo or in vitro protein aging experiments.