Deamidation and related problems in structural analysis of peptides and proteins
Sargaeva, Nadezda P
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Electron capture dissociation (ECD) and electron transfer dissociation (ETD) can generate unique fragments and preserve post-translational modifications (PTMs), enabling their detection in biological samples. They have been used to differentiate isomeric aspartic (Asp) and isoaspartic acids (isoAsp) produced upon non-enzymatic deamidation of asparagine (Asn) -- a frequently occurring PTM. IsoAsp formation was detected in amyloid-β (Aβ) peptides in the specimens of Alzheimer's disease (AD) patients, and is a potential biomarker for AD if it can be detected early in biofluids of live individuals. Synthetic isoAsp-containing Aβ fragments were studied using ECD to test the method's applicability. IsoAsp-7 and -23 were detected in top-down analysis of the 4.5kDa Aβ42 protein and in Aβ17-28 peptide. Further, a related method, electron ionization/impact dissociation (EID), was successfully applied to Asp/isoAsp differentiation for the first time. High-performance liquid chromatography (HPLC) is a powerful technique for the separation of complex mixtures. HPLC separation of Asp- and isoAsp-containing peptides revealed inconsistent elution orders, especially when isoAsp was located at the N-terminus, requiring ECD for identification. New diagnostic fragments were proposed for N-terminal isoAsp based on the ECD and ETD results. Challenges in detection of such fragments were improved by supplemental activation and chemical modifications. Furthermore, a model for retention time prediction was applied to isoAsp-containing peptides and suggested for their improved identification in HPLC-MS/MS approach. IsoAsp is a 13-amino acid, which distinctively contains an additional methylene group in the backbone, forming a Cα-Cβ bond. Cleavage of this bond provides diagnostic fragments for isoAsp by ECD. The same was proposed for other β-amino acids. However, the Cα-Cβ bond cleavages were rare due to the instability of the Cβ radical. Alternatively, in-source decay (ISD) fragmentation during matrixassisted laser desorption/ionization (MALDI) process can produce abundant ECD-like fragmentation. It was proposed that use of hydrogen-accepting matrices may lead to Cα-Cβ bond cleavage in β-amino acids, because the resulting radical would be stabilized by the carbonyl group. To test this, β-amino acid-containing peptides were analyzed by MALDI-ISD using 5-nitrosalicylic acid matrix. The Cα-Cβ bond cleavages were observed. Overall, new and improved methods were implemented allowing better characterization and differentiation of β-amino acids.
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