Inertial Agitation in an NMR Magnet: Real-Time Monitoring of Protein Aggregation at High Resolution

We present a simple and efficient method to agitate NMR samples and thereby induce protein aggregation directly inside an NMR magnet. In this approach, termed inertia NMR, the rotational inertia of an eccentric floater within the sample tube generates agitation during acceleration and deceleration of tube spinning. This method enabled the aggregation of multiple disease-associated proteins under near-physiological conditions while allowing real-time monitoring by NMR spectroscopy. By combining inertia NMR with 2D NMR, we followed the aggregation of an α-synuclein (αS) isoform at high resolution. In addition, isotope editing/filtering in the presence of inertial agitation allowed individual tracking of protein coaggregation. Remarkably, αS aggregation was accelerated by its splicing isoform and even more strongly by the C-terminal domain of TDP-43 (TDP-43 CTD), underscoring the role of cross-seeding in synucleinopathies. Furthermore, inertia NMR demonstrated diagnostic potential by discriminating Parkinson's disease from multiple system atrophy using patient-derived seeds. This method provides high-resolution insight into protein aggregation and offers a general, instrument-free strategy to accelerate chemical processes inside an NMR magnet through efficient sample mixing.