High-quality 13C-detected structural analysis of mass-limited amyloid samples using a CPMAS CryoProbe and moderate magnetic fields.

Solid-state NMR (SSNMR) of biomolecules typically requires several milligrams of sample to achieve sufficient sensitivity for multidimensional experiments, especially when relying on 13C detection. Recent developments in fast magic-angle spinning (MAS) and 1H-detected methods have enabled the use of submilligram samples in reduced-diameter rotors, but these approaches demand advanced hardware and often suffer from limited 1H chemical shift dispersion. Here, we demonstrate that a CPMAS CryoProbe enables the acquisition of high-quality 13C-detected 2D and 3D spectra from just ∼1.5 mg of uniformly labeled amyloid fibrils packed in a standard 3.2 mm rotor. As a proof of concept, we apply this approach to RIPK3, a key protein in immune signaling that forms functional amyloid assemblies. Using standard 3D experiments (NCACX and NCOCX), we obtain 13C and 15N backbone assignments and secondary structure information, despite the limited sample quantity and the use of only moderate magnetic fields. These findings highlight the potential of CPMAS CryoProbes to shift the paradigm in mass-limited SSNMR studies, from relying exclusively on 1H-detection and fast MAS to reembracing 13C-detected strategies.