Probing early α-Synuclein oligomers: Insights into aggregation pathways of NACore and preNAC segments probed by Trapped Ion-Mobility Mass Spectrometry and Fluorescence Spectroscopy

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 (N2CCSTIMS) 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.