Determining the Location of the Alpha-Synuclein Dimer Interface using Native Top-Down Fragmentation and Isotope Depletion-Mass Spectrometry

Alpha-synuclein (αSyn), a 140-residue intrinsically disordered protein, comprises the primary proteinaceous component of pathology-associated Lewy body inclusions in Parkinson’s disease (PD). Due to its association with PD αSyn is studied extensively; however the endogenous structure and physiological roles of this protein are yet to be fully understood. Here, ion mobility-mass spectrometry and native top-down electron capture dissociation fragmentation have been used to elucidate the structural properties associated with a stable, naturally-occurring dimeric species of αSyn. This stable dimer appears in both wild-type (WT) αSyn and the PD-associated variant A53E. Furthermore, we integrated a novel method for generating isotopically depleted protein into our native top-down workflow. Isotope depletion increases signal-to-noise ratio and reduces the spectral complexity of fragmentation data, enabling the monoisotopic peak of low abundant fragment ions to be observed. This enables the accurate and confident assignment of fragments unique to the αSyn dimer to be assigned, and structural information about this species to be inferred. Using this approach, we were able to identify fragments unique to the dimer, which demonstrates a C-terminal to C-terminal interaction between the monomer subunits. The approach in this study holds promise for further investigation into the structural properties of endogenous multimeric species of αSyn. The data relates to the following publication: Kiani Jeacock, Alexandre Chappard, Kelly J. Gallagher, C. Logan Mackay, David P. A. Kilgour, Mathew H. Horrocks, Tilo Kunath and David J. Clarke. 'Determining the Location of the Alpha-Synuclein Dimer Interface using Native Top-Down Fragmentation and Isotope Depletion-Mass Spectrometry.' J. Am. Soc Mass Spec. (in submission).