Discrimination of Aspartic and Isoaspartic Acid Residues in Peptides by Tandem Mass Spectrometry with Hydrogen Attachment Dissociation

Long-lived proteins undergo chemical modifications that can cause age-related diseases. Among these chemical modifications, isomerization is the most difficult to identify. Isomerization often occurs at the aspartic acid (Asp) residues. In this study, we used tandem mass spectrometry equipped with a newly developed ion activation method, hydrogen attachment dissociation (HAD), to analyze peptides containing Asp isomers. Although HAD preferentially produces [c + 2H] and [z + 2H] via N-Cα bond cleavage, [c + 58 + 2H] and [z - 58 + 2H] originate from the fragmentation of the isoAsp residue. Notably, [c + 58 + 2H] and [z - 58 + 2H] could be used as diagnostic fragment ions for the isoAsp residue because these fragment ions did not originate from the Asp residue. The detailed fragmentation mechanism was investigated by computational analysis using density functional theory. According to the results, hydrogen attachment to the carbonyl oxygen in the isoAsp residue results in the Cα-Cβ bond cleavage. The experimental and theoretical joint study indicates that the present method allows us to discriminate Asp and isoAsp residues, including site identification of the isoAsp residue. Moreover, we demonstrated that the molar ratio of peptide isomers in the mixture could be estimated from their fragment ion abundance. Therefore, tandem mass spectrometry with HAD is a useful method for the rapid discrimination and semiquantitative analysis of peptides containing isoAsp residues.