Amyloid β peptides are the major components of the vascular and plaque amyloid filaments in individuals with Alzheimer’s disease (AD). Although it is still unclear what initiates the disease, isomerization of aspartic acid residues in Aβ peptides is directly related to the pathology of AD. The detection of isomerization products is analytically challenging due to their similar chemical properties and identical molecular mass. Different methods have been applied to differentiate and quantify the isomers, including immunology, chromatography and mass spectrometry. Typically, those methods require comparative analysis with the standard peptides and involve many sample preparation steps. To understand the role of Aβ isomerization in AD progression, a fast, simple, accurate, and reproducible method is necessary. In this work, Electron Capture Dissociation (ECD) Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR MS) was applied to detect isomerization in Aβ peptides. ECD generated diagnostic fragment ions for the two isomers of Aβ17-28: [M+2H-60]•+ and z6•-44 when aspartic acid and z6•-57 when isoaspartic acid were present. Additionally, the zn-57 diagnostic ion was also observed in the Electron Ionization Dissociation (EID) spectra of the modified Aβ17-28 fragment. ECD was further applied towards Aβ1-40 and A1-β42. The diagnostic ion c6+57 was observed in the ECD spectra of the A1-β42 peptide demonstrating isomerization at residue 7. In conclusion, both ECD and EID can clearly determine the presence and the position of isoaspartic acid residues in Amyloid β peptides. The next step, therefore, is to apply this method to analyze samples of Alzheimer’s patients or healthy individuals in order to generate a better understanding of the disease.

Formation of isoaspartic acid (isoAsp) is a common modification of aspartic acid (Asp) or asparagine (Asn) residue in proteins. Differentiation of isoAsp and Asp residues is a challenging task owing to their similar properties and identical molecular mass. It was recently shown that they can be differentiated using ion-electron or ion-ion interaction fragmentation methods (ExD), as these methods provide diagnostic fragments c + 57 and z• − 57 specific to the isoAsp residue. To date, however, the presence of such fragments has not been explored on peptides with an N-terminal isoAsp residue. To address this question, several N-terminal isoAsp-containing peptides were analyzed using ExD methods alone or combined with chromatography. A diagnostic fragment [M + 2H − 74]+• was observed for the doubly charged precursor ions with N-terminal isoAsp residues. For some peptides, identification of the N-terminal isoAsp residue was challenging due to the low diagnostic ion peak intensity and the presence of interfering peaks. Supplemental activation was used to improve diagnostic ion detection. Further, N-terminal acetylation was offered as a means to overcome the interference problem by shifting the diagnostic fragment peak to [M + 2H − 116]+•.

Ion-electron reaction based fragmentation methods (ExD) in tandem mass spectrometry (MS), such as Electron Capture Dissociation (ECD) and Electron Transfer Dissociation (ETD) represent a powerful tool for biological analysis ExD methods have been used to differentiate the presence of the isoaspartate (isoAsp) from the aspartate (Asp) in peptides and proteins. IsoAsp is a β3-type amino acid that has an additional methylene group in the backbone, forming a Cα-Cβ bond within the polypeptide chain. Cleavage of this bond provides specific fragments that allow differentiation of the isomers. The presence of a Cα-Cβ bond within the backbone is unique to β-amino acids, suggesting a similar application of ExD toward the analysis of peptides containing other β-type amino acids. In the current study, ECD and ETD analysis of several β-amino acid containing peptides was performed. It was found that N-Cβ and Cα-Cβ bond cleavages were rare, providing few c and z• type fragments, which was attributed to the instability of the Cβ radical. Instead, the electron capture resulted primarily in the formation of a• and y fragments, representing an alternative fragmentation pathway, likely initiated by the electron capture at a backbone amide nitrogen protonation site within the beta amino acid residues.

Deamidation of asparagine and spontaneous isomerization of aspartic acid in proteins and peptides occur frequently. These modifications result in a mixture of peptide variants containing all three residues in the sequences. Identification and isomer quantification for these systems are challenging tasks for tandem mass spectrometry commonly utilized in protein analysis. Chromatographic data provide a set of sequence-specific information complementary to mass spectrometry. In order to compare measured retention times (RTs) with those calculated from the sequences derived from protein databases, it is necessary to develop chromatographic models and tools allowing the prediction of RT and elution order for peptides with modified residues. In this work we extended recently introduced critical liquid chromatography of biomacromolecule model for prediction of RTs for peptides containing asparagines, aspartic acid, and isoaspartic acid residues.

Deamidation and the subsequent formation of isoaspartic acid (isoAsp) are common modifications of asparagine (Asn) residues in proteins. Differentiation of isoAsp and Asp residues is a challenging task owing to their similar chemical properties and identical molecular mass. Recent studies showed that they can be differentiated using electron capture dissociation (ECD) which generates diagnostic fragments c′+57 and z•−57 specific to the isoAsp residue. However, the ECD approach is only applicable towards multiply charged precursor ions and generally does not work for β-amino acids other than isoAsp. In this study, the potential of in-source decay (ISD) in characterization of isoAsp and other β-amino acids was explored. For isoAsp-containing peptides, ISD with a conventional hydrogen-donating matrix produced ECD-like, c′+57 and z•−57 diagnostic ions, even for singly charged precursor ions. For other β-amino acids, a hydrogen-accepting matrix was used to induce formation of site-specific a-14 ions from a synthetic β-analogue of substance P. These results indicated that ISD can be broadly applied for β-peptide characterization.