Differentiating ?- and ?-aspartic acids by electrospray ionization and low-energy tandem mass spectrometry

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Abstract
Spectra obtained by low‐energy electrospray ionization tandem mass spectrometry (ESI‐MS/MS) of 34 peptides containing aspartic acids at position n were studied and unambiguously differentiated. β‐Aspartic acid yields an internal rearrangement similar to that of the C‐terminal rearrangements of protonated and cationized peptides. As a result of this rearrangement, two different ions containing the N‐ and the C‐terminal ends of the original peptide are formed, namely, the b n − 1 + H2O and y″ℓ − n + 1 − 46 ions, respectively, where ℓ is the number of amino acid residues in the peptide. The structure suggested for the y″ℓ − n + 1 − 46 ion is identical to that proposed for the vn ions observed upon high‐energy collision‐induced dissociation (CID) experiments. The intensity of these ions in the low‐energy MS/MS spectra is greatly influenced by the presence and position of basic amino acids within the sequences. Peptides with a basic amino acid residue at position n − 1 with respect to the β‐aspartic acid yield very intense b n−1 + H2O ions, while the y″ℓ − n + 1 − 46 ion was observed mostly in tryptic peptides. Comparison between the high‐ and low‐energy MS/MS spectra of several isopeptides suggests that a metastable fragmentation process is the main contributor to this rearrangement, whereas for long peptides (40 AA) CID plays a more important role. We also found that α‐aspartic acid containing peptides yield the normal immonium ion at 88 Da, while peptides containing β‐aspartic acid yield an ion at m/z 70, and a mechanism to explain this phenomenon is proposed. Derivatizing isopeptides to form quaternary amines, and performing MS/MS on the sodium adducts of isopeptides, both improve the relative intensity of the b n + 1 + H2O ions. Based on the above findings, it was possible to determine the isomerization sites of two aged recombinant growth proteins. Copyright © 2000 John Wiley & Sons, Ltd.