Naturally occurring D-amino acid substitution, also known as amino acid D-isomerization, has been observed in many disease-associated peptides and proteins, including amyloid beta (Aβ), one of the putative biomarkers and drug targets for Alzheimer’s disease (AD).  Aβ is of significant interest due to the prevalence of post-translational D-isomerization in AD brain samples.  While many prior reports have described technical advancements associated with the chiral discrimination and separation of D-amino acid containing peptides, there remains a dearth of tools capable of targeting Aβ42 stereochemistry. Ion mobility-mass spectrometry (IM-MS) has increasingly become an important alternative for the chiral separation of Aβ stereoisomers. IM-MS offers high analytical speed, low sample consumption and the ability to resolve small structural differences in peptide analytes, driven by recent technological advancements in IM-MS.  We recently reported a multi-dimensional IM-MS (md-IM-MS)-based structural analysis strategy to facilitate the study of the chiral effects on monomer structure, oligomeric propensity and receptor binding for Aβ fragments.<br/>To systematically probe the chiral effects in full length, bioactive Aβ42 including the differential roles of the N terminal and core regions, we herein demonstrate an improved md-IM-MS approach for the systematic study of the structural consequences of domain-specific Asp/Ser D-isomerization within Aβ42. Cytotoxicity of Aβ isoforms in the context of neuronal cell lines were examined using a series of custom-synthesized peptides, which were also subjected to structural analysis using an improved md-IM-MS platform based on composite IM measurements across multiple instrument platforms. Analytical challenges facing the MS characterization of full length Aβ42 have been successfully tackled through an improved sample preparation workflow. Moreover, IM-MS-guided replica exchange MD (REMD) simulations were employed to construct and refine the 3D structures of different Aβ42 stereoisomers. Our analysis reveals that co-D-isomerization at Asp and Ser residues of Aβ42 in both the N-terminal and core regions effectively and synergistically rescues its cytotoxicity, seemingly related to the differential and domain-specific changes in both overall and local 3D shapes, among which structural compaction exhibits the most significant effect.

Chirality regulated amyloid beta self-assembly and receptor recognition probed by multi-dimensional ion mobility mass spectrometry

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ACS Fall 2022

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