Fragmentation and aggregation of physiological and parkinson- synucleins revealed by ion mobility and affinity- mass spectrometry

Abstract

A large variety of cellular processes are based on the formation of supramolecular protein assemblies. Several diseases, previously thought to be unrelated, are characterised by “misfolded” protein aggregates. Structures and reaction pathways of pathophysiological aggregates are only poorly characterised at present. “Soft-ionisation” mass spectrometry (MS), such as HPLC-MS, is often unsuitable to the analysis of reactions and intermediates in aggregation. Recently, ion mobility- MS (IM-MS) is emerging as a powerful tool for analysis of protein aggregation due to its concentration-independent gas phase separation capability. Applications of IM-MS to the in vitro oligomerization- aggregation of α-synuclein (αSyn), a key protein for Parkinson’s disease, provided the first identification of a specific autoproteolytic fragmentation, particularly a highly aggregation-prone fragment by cleavage at the triplett, VVT(70-72) of the central aggregation domain [1]. The corresponding recombinant αSyn(72-140) fragment showed substantially faster aggregation and high neurotoxicity compared to the intact protein. The recent development of online bioaffinity-MS [2] enabled first direct structural studies in vivo, e.g. from brain homogenate. Applications of affinity-MS will be discussed for the characterization of oligomers and assemblies in vivo. Moreover, specific mutations of the central (70-72) triplett in synucleins provided a breakthrough by mutation of VFS(70-72) from ß-Synuclein into the αSyn sequence, that completely abolished neurotoxic aggregation. These results suggest ion mobility- MS and affinity-MS as powerful tools for the elucidation of structures and intermediates of polypeptide aggregation, providing a basis for the molecular study of oligomerization- aggregation pathways, and the design of specific inhibitors of aggregation.