Differentiating N-Terminal Aspartic and Isoaspartic Acid Residues in Peptides

Abstract

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]+•.

Quantification of N-terminal amyloid-β isoforms reveals isomers are the most abundant form of the amyloid-β peptide in sporadic Alzheimer’s disease

“… 13 Previous reports characterizing stereoisomers of synthetic Aβ have particularly demonstrated that isomerization at position Asp-1 and Asp-7 is frequent. 23 , 38 , 52 However, neither the extent of isomerization of Asp-1 and Asp-7 residues in Aβ 1 – 15, Aβ 2 – 15 , Aβ 3Glu-15 and Aβ 4 – 15 have been directly measured in human brain nor has a systematic study been conducted using ion mobility to determine the effect of isomerization on the structure of these peptides in the gas phase. We postulated that the orthogonality of the online LC and drift tube ion mobility separations (DT-IMS) would provide the required analytical resolution, even if at modest R ∼ 50, to resolve the N-terminal Aβ isomers/racemers derived from Alzheimer’s disease brains and improve the detection/quantification limits of these complex biological samples.…”

Aspartic Acid Isomerization Characterized by High Definition Mass Spectrometry Significantly Alters the Bioactivity of a Novel Toxin from Poecilotheria

“…ETD utilized an electron transfer in gas-phase ion-ion chemistry, causing rearrangement with cleavages of the N-C α backbone referred to as c and z ions [60]. While the fragment ratios seen in CID differ suggesting structural discontinuity between the peptides, ETD cleaves the isomers of Asp producing "reporter" ions in isoAsp not seen in Asp [61]. These reporter ions are a result of an alternate cleavage due to presence of the extra methylene group in the peptide backbone of isoAsp (Figure 7).…”

Recent advances in mass spectrometric analysis of protein deamidation

“…Two major isozymes of PIMT have been isolated from human erythrocytes via isoelectric focusing (Ota et al, ). Although PIMT minimizes accumulation of potentially detrimental isoAsp in aged proteins, the repair process is unable to restore Asn residues, hence the effects of deamidation are partly irreversible (Yang & Zubarev, ; Sargaeva et al, ). PIMT expression appears to differ between tissues, and in rat brain the enzyme is slowly upregulated after birth and remains highly expressed throughout natural aging (Mizobuchi et al, ).…”