Probing Early α-Synuclein Oligomers: Insights Into Aggregation Pathways of NACore and preNAC Segments Probed by...

Misfolding and aggregation of α-Synuclein (α-Syn) play a central role in Parkinson's disease (PD), with oligomeric intermediates implicated as key toxic species. Here, we investigate the aggregation of two α-Syn segments, the NACore (₆₈GAVVTGVTAVA₇₈, WT-PD1) and the preNAC region (₄₇GVVHGVATVA₅₆, WT-PD2), using high-resolution trapped ion-mobility mass spectrometry (TIMS-Qq-ToF) and Thioflavin T fluorescence spectroscopy. The NACore is the minimal sequence required for α-Syn aggregation, whereas most mutations affecting the onset of PD appear to be in the preNAC region, therefore modulating aggregation dynamics and toxicity. Our results demonstrate that TIMS-Qq-ToF effectively resolves and identifies oligomeric species, revealing distinct aggregation pathways for both peptide segments. Fluorescence assays confirm differences in aggregation kinetics and morphology, highlighting the different oligomer formation pathways observed with ion-mobility mass spectrometry. Despite the oligomer-preserving nature of the TIMS itself, ion transmission remains too harsh for the fragile oligomers, leading to fragmentation of fragile non-covalent assemblies. However, collisional cross-section (^N2CCS_TIMS) values support the presence of large oligomers. This study highlights the potential of TIMS-Qq-ToF for mapping α-Syn aggregation and underscores the need for optimised, soft ion transmission to better preserve fragile transient intermediates, ultimately contributing to a deeper understanding of PD. SUMMARY: Understanding early-stage protein aggregation is essential for unravelling the molecular mechanisms of Parkinson's disease, a neurodegenerative disorder that currently lacks effective therapeutic solutions. This study employs high-resolution Trapped Ion Mobility Mass Spectrometry combined with fluorescence spectroscopy to elucidate the oligomerisation of two α-synuclein segments, revealing distinct aggregation pathways and associated structural characteristics. This study underscores the value of peptide models in advancing our understanding of protein aggregation behaviour. This multifaceted approach provides detailed structural insights into the unexplored, transient early-stage oligomers of key α-Syn segments, contributing to a deeper understanding of aggregation mechanisms and providing valuable insights for therapeutic strategies against Parkinson's disease.