Uncovering molecular details of protein "misfolding - aggregation" using affinity- and ion mobility- mass spectrometry - Physiological and “Parkinson”- Synucleins
Michael Przybylski, Department of Chemistry and Steinbeis Research Center for Biopolymer Chemistry & Biomolecular Mass Spectrometry, University of Konstanz
A large variety of cellular processes are based on the formation and dynamics of multi- and supramolecular protein assemblies, and several diseases, previously thought to be unrelated, such as cancer and neurodegenerative diseases, are characterised by “misfolded” protein aggregates.
Chemical structures and reaction pathways of pathophysiological aggregates are only poorly characterised at present. “Soft-ionisation” mass spectrometry (MS), such as HPLC-electrospray-MS, is often unsuitable to direct analysis of reaction pathways and intermediates in aggregation. Recently, ion mobility- MS (IM-MS) has been emerging as a new tool for analysis of protein aggregation due to its concentration-independent gas phase separation capability.
First applications of IM-MS to the in vitro oligomerization of α-synuclein (αSyn) and ß-amyloid (Aß), key proteins for Parkinson’s disease and Alzheimer’s disease, enabled the identification of hitherto unknown degradation and aggregation products. Time- dependent studies of the in vitro oligomerization- aggregation of αSyn provided the first identification of a specific autoproteolytic fragmentation, particularly a highly aggregation-prone fragment by cleavage at V71/T72 in the ß-breaking triplett VVT(70-72) in 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 combined, online bioaffinity-MS methods [2] enabled first direct (“top-down”) structural studies in vivo, such as from brain homogenate. Applications of affinity-MS will be discussed using epitope-specific αSyn- antibodies [3] for the characterization of oligomers and interactions in vivo.
Most recently, specific mutations of the central (70-72) triplett in synucleins, and affinity-MS provided breakthrough results, such as mutation of the VFS(70-72) triplett from physiological ßSyn into αSyn completely abolished neurotoxic aggregation [4]. These results suggest ion mobility- MS and affinity-MS as powerful tools for the molecular elucidation of structures and intermediates of polypeptide aggregation, providing a basis for (i), the molecular study of oligomerizationaggregation pathways, and (ii), the design of peptides capable of inhibiting or modifying aggregation.
[1] Vlad, C. et al., (2011) ChemBiochem. 12, 2740-2744.
[2] Dragusanu, M., et al. (2010) J. Am. Soc. Mass Spectrom. 21, 1643-1648.
[3] McLaurin, J., et al. (2002) Nature Med. 8: 1263-1269
[4] Przybylski, M. et al. (2013) submitted for publication.
