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

Abstract

α-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 wildtype (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.

CIUSuite 3: Next-Generation CCS Calibration and Automated Data Analysis Tools for Gas-Phase Protein Unfolding Data

“…With growing interest and application of CIU, stability shift analyses can become less intuitive depending on the system being studied. For instance, some charge states of α-synuclein have been observed to exhibit parallel CIU features across multiple collision voltages, complicating robust stability comparisons . In addition, CIU of nucleic acids, especially RNAs, typically undergo slight compaction transitions prior to unfolding during CIU, necessitating nonstandard approaches for the evaluation of CIU50 values .…”

Cyclic Ion Mobility-Mass Spectrometry and Tandem Collision Induced Unfolding for Quantification of Elusive Protein Biomarkers

“…Prior work has demonstrated that CIU can uncover partially folded conformational intermediates that are unique to isoforms and structural states that are overlooked by other MS methods . For instance, CIU has been used to capture subtle differences in protein structure due to disease-associated sequence variation, − disulfide bonding, post-translational modifications, and ligand binding . More recently, CIU has been used as a tool to quantify large (66–150 kDa) proteins exhibiting similar molecular weights and sequences. , Despite the ability of CIU to differentiate and quantify subtly different protein variants, CIU has yet to be used to quantify disease-associated small (<15 kDa) biomarkers present in biological matrices.…”

Cyclic Ion Mobility-Mass Spectrometry and Tandem Collision Induced Unfolding for Quantification of Elusive Protein Biomarkers

“…Prior work has demonstrated that CIU can uncover partially folded conformational intermediates which are unique to isoforms and structural states that are overlooked by other MS methods. 61 For instance, CIU has been used to capture subtle differences in protein structure due to disease-associated sequence variation, [62][63][64] disulfide bonding, 65 posttranslational modifications, 66 and ligand binding. 67 More recently, CIU has been used as a tool to quantify large (66-150 kDa) proteins exhibiting similar molecular weights and sequences.…”