Neurodegenerative diseases share complex molecular mechanisms, including protein misfolding, oligomerization, and aggregation.[1] A prominent example is Amyotrophic Lateral Sclerosis (ALS), a fatal neuromuscular disorder characterized by the accumulation of both soluble oligomers and insoluble aggregates of Cu/Zn Superoxide Dismutase (SOD1) bearing non-native conformations, resulting from over 120 distinct gene mutations.[2] Despite the central role of early aggregation in ALS pathology, the underlying mechanisms remain poorly understood, primarily due to the lack of analytical techniques capable of capturing the dynamic processes of SOD1 misfolding and oligomerization. In this study, bovine SOD1 was used to establish an assay that combines temperature-controlled nano-electrospray ionization (TC-nESI) with ion mobility-mass spectrometry (IM-MS), enabling real-time structural and conformational analysis of accelerated early aggregation events.

Native bovine SOD1 exists as a homodimer, with each subunit coordinating one Cu(II) and one Zn(II) ion. As metal loss is known to destabilize SOD1 and promote aggregation, the study focused on holo-SOD1 and apo-SOD1 (metal-free) variants. Thermal unfolding experiments were conducted using a custom-built TC-nESI source coupled to cyclic IMS, to examine the effects of metal-dependent structural stability under different solution heating rates (1.0 and 0.3 °C/min). For holo-SOD1, both fast and slow heating led to dimer dissociation into monomers of variable folds; however, only slow heating resulted in sufficient accumulation of misfolded monomers to enable enhanced and fast oligomer formation. Interestingly, slow thermal denaturation of apo-SOD1 revealed trimers and misfolded monomers as intermediates in subsequent apo-oligomer assembly. Compared to holo-SOD1, apo-SOD1 produced more compact and abundant oligomers, following a more gradual assembly process, while collision cross section (CCS) calculations indicated that both variants follow isotropic oligomer growth pathways. Surface-induced dissociation MS/MS (SID-MS/MS) experiments unveiled oligomerization assembly mechanisms, with primarily monomeric and dimeric subunits acting as building blocks in apo-derived oligomers. Additional insights from limited proteolysis and heat-induced fragmentation identified loops V, VI, and VII near the C-terminal region as particularly labile in misfolded apo-SOD1 monomers. Collectively, these findings highlight the power of TC-nESI combined with IM-MS to simultaneously resolve misfolding transitions, identify key intermediates, and characterize the architecture of co-populated oligomeric species, crucial for elucidating protein dynamics in neurodegeneration.

[1] C. Soto, S. Pritzkow, Nature Neuroscience, 2018, 21, 1332–1340.

[2] L. McAlary, Y. L. Chew, J. S. Lum, N. J. Geraghty, J. J. Yerbury, N. R. Cashman, Frontiers in Cellular Neuroscience, 2020, 14, 339.