Co-existence of two different α-synuclein oligomers with different core structures determined by hydrogen/deuterium exchange mass spectrometry.

Neurodegenerative disorders are characterized by the formation of protein oligomers and amyloid fibrils, which in the case of Parkinson's disease involves the protein α-synuclein (αSN). Cytotoxicity is mainly associated with the oligomeric species, but we still know little about their assembly and structure. Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is used to analyze oligomers formed by wild-type (wt) αSN and also three familial αSN mutants (A30P, E46K, and A53T). All four variants show co-existence of two different oligomers. The backbone amides of oligomer type I are protected from exchange with D2 O until they dissociate into monomeric αSN by EX1 exchange kinetics. Fewer residues are protected against exchange in oligomer type II, but this type does not revert to αSN monomers. Both oligomers are protected in the core sequence Y39-A89. Based on incubation studies, oligomer type I appears to form straight fibrils, while oligomer type II forms amorphous clusters that do not directly contribute to the fibrillation process.

Electron capture dissociation proceeds with a low degree of intramolecular migration of peptide amide hydrogens.

Hydrogen (1H/2H) exchange combined with mass spectrometry (HX-MS) has become a recognized method for the analysis of protein structural dynamics. Presently, the incorporated deuterons are typically localized by enzymatic cleavage of the labeled proteins and single residue resolution is normally only obtained for a few residues. Determination of site-specific deuterium levels by gas-phase fragmentation in tandem mass spectrometers would greatly increase the applicability of the HX-MS method. The biggest obstacle in achieving this goal is the intramolecular hydrogen migration (i.e., hydrogen scrambling) that occurs during vibrational excitation of gas-phase ions. Unlike traditional collisional ion activation, electron capture dissociation (ECD) is not associated with substantial vibrational excitation. We investigated the extent of intramolecular backbone amide hydrogen (1H/2H) migration upon ECD using peptides with a unique selective deuterium incorporation. Our results show that only limited amide hydrogen migration occurs upon ECD, provided that vibrational excitation prior to the electron capture event is minimized. Peptide ions that are excessively vibrationally excited in the electrospray ion source by, e.g., high declustering potentials or during precursor ion selection (via sideband excitation) in the external linear quadrupole ion trap undergo nearly complete hydrogen (1H/2H) scrambling. Similarly, collision-induced dissociation (CID) in the external linear quadrupole ion trap results in complete or extensive hydrogen (1H/2H) scrambling. This precludes the use of CID as a method to obtain site-specific information from proteins that are labeled in solution-phase 1H/2H exchange experiments. In contrast, the deuteration levels of the c- and z-fragment ions generated from ECD closely mimic the known solution deuteration pattern of the selectively labeled peptides. This excellent correlation between the results obtained from gas phase and solution suggests that ECD holds great promise as a general method to obtain single residue resolution in proteins from solution 1H/2H exchange experiments.