Intrinsically disordered proteins (IDPs) contain highly flexible regions, which bind to different protein partners, thereby helping to maintain proper cell function. α-Synuclein is an IDP and can form aggregate species leading to Parkinson’s disease. Learning how protein structure relates to aggregate formation is critical in understanding the disease mechanism. α-Synuclein forms various structures in solution, which relax into multiple gas-phase conformers. This information is preserved by native top-down mass spectrometry (MS), unlike a bottom-up crosslinking approach. The wide distribution of charge states observed for α-Synuclein is reflective of its structural heterogeneity. In this study, native protein crosslinking, ion mobility separation and tandem MS were combined to crosslink reactive side chain residues from distinct charge states, thus providing residue-level information and distance constraints for individual conformers. The crosslinking strategies included the use of a panel of different electrostatic and covalent reagents to target protonated side chains in the gas phase and primary amines in solution respectively. Unique crosslinked sites were obtained in solution and in the gas phase, demonstrating that reagents of different lengths and flexibility target distinct regions of α-Synuclein. The identified crosslinked residues are currently being used as distance constraints to filter protein models generated by molecular dynamics (MD) simulations. Overall, native protein crosslinking, followed by ion mobility and tandem MS analysis, provides a unique approach in studying the structural diversity of an IDP such as α-Synuclein.

Leveraging native protein crosslinking and ion mobility-mass spectrometry to capture the structural variety of α-Synuclein

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