MS strategies to add a structural dimension in conversion-induced aggregation assays for the early detection of pathogenic a-Synuclein strains – MS-QuIC

Synucleinopathies are characterized by the accumulation of a-Synuclein fibrils in tissues. Fibril precursors are misfolded proteins that can exist as different structurally distinct strains, potentially associated to different diseases. Real time quaking induced conversion (RT-QuIC) is an efficient method to detect the presence of the precursors before the apparition of fibrils, building on their ability to convert normal proteins to pathogenic forms. In RT-QuIC assays, a medium containing normal proteins is seeded with a low quantity of pathogenic species, and the apparition of fibrils is monitored in real time using specific fluorescent probes, like thioflavin T (ThT). However, RT-QuIC only measures the formation of aggregates, but does not give information of their structure. In particular, it is not clear how and by which mechanism the RT-QuIC response is affected by the structure of the initial pathogenic species in the seed. The MS-QuIC project aims at gaining structural insight in the RT-QuIC process in order to better exploit RT-QuIC data. To this end we propose an original combination of mass spectrometry-based techniques that will allow to characterize the aggregating species from the smallest oligomers to the fibrils. Namely, ion mobility mass spectrometry measurements (IMSMS) will allow to characterize the structure and morphology of the small oligomers and their complexes with ThT, while charge detection mass spectrometry (CDMS) measurements will be used for fibrils and protobibrils, whose morphology will be studied by electron microscopy. Both CDMS and IMSMS will be performed on samples collected at different steps of RT-QuIC experiments seeded with amplification material obtained from different clinical strains of a-Synuclein. Correlation with RT-QuIC will be allowed through in-source fluorescence measurements online the CDMS and IMSMS instruments. Beyond RT-QuIC, the proposed experimental strategy will be applicable to other aggregation processes.