IR Action Spectroscopy of Mass- and Mobility-Selected α-Synuclein Segments: Structure Identification in Early-Stage...

Parkinson's disease (PD) is associated with protein misfolding and aggregation of α-Synuclein (α-Syn), a process central to its pathology. Understanding the early structural transitions of α-Syn is crucial, yet detailed insights into oligomeric intermediates remain elusive. Using the novel Photo-Synapt spectrometer, which integrates ion mobility mass spectrometry (IM-MS) with infrared multiple-photon dissociation (IRMPD) spectroscopy, we investigated the secondary structures of key α-Syn peptide segments, namely, WT-PD1 (NACore) and WT-PD2 (pre-NAC). This unique approach enabled conformationally selective analysis, allowing peptide monomers and oligomers to be separated based on both mass and mobility. Despite sequence similarities, these peptides exhibit distinct monomeric structures and aggregation behavior, leading to different oligomer assemblies with unique IR signatures. By analyzing the amide A region (3100-3500 cm^-1), we provide structural insight into α-Syn oligomer structure, including the resolution of parallel versus antiparallel β-sheets and the identification of non-β motifs such as γ-turns. We further examined the G51D mutation from the pre-NAC region (G51D-PD2), which induces a more compact structure and stabilizes oligomers, leading to distinct IR spectral features. Combining IM-MS and IRMPD spectroscopy within a single instrument provides a novel structural readout for early-stage peptide aggregation. This approach highlights the high sensitivity of sequence-dependent structural transitions in α-Synuclein oligomers, providing insight into the mechanistic pathways of sequence-dependent folding relevant to disease.