Hyphenated Mass Spectrometry-Based Strategies for Characterizing Oligomers of α‑Synuclein in Parkinson's Disease.

The pathological aggregation of α-synuclein (α-syn), an intrinsically disordered protein that regulates synaptic vesicle trafficking in the brain, is a defining molecular feature in Parkinson's disease (PD). Early oligomeric assemblies are widely considered the most neurotoxic species, yet their structural features remain unexplored due to their low abundance, transient, and fragile nature. Here, we establish a trapped ion mobility-mass spectrometry (TIMS-MS) strategy that enables direct detection and structural interrogation of these oligomers. Low-flow size exclusion chromatography provides MS-compatible desalting and reveals broad ensemble-level differences between fresh and aggregated α-syn. Native TIMS-MS measurements uniquely probe numerous higher-order oligomers, thereby revealing a reduced conformational heterogeneity with increasing oligomer size, supporting a linear β-strand-type assembly pathway. These findings show that oligomerization drives a transition from dynamic monomers to increasingly ordered intermediates. This progressive rigidification is relevant to models in which α-syn oligomerization alters normal membrane engagement during PD-associated aggregation.