Cross-Linking and Covalent Labeling Mass Spectrometry Reveal Proteoform-Driven Conformational Changes in Alpha Synuclein

Phosphorylation of serine 129 (pS129) in the intrinsically disordered protein alpha synuclein has long been associated with neurodegenerative diseases. In recent years, the functional relevance of pS129 has been uncovered by electrophysiology, immunoprecipitation, and proteomics, revealing its intricate connection to neurotransmitter release and synaptic vesicle (SV) cycling. In this study, chemical cross-linking and covalent labeling of alpha synuclein and pS129, as well as an additional form encountered in the brain, oxidized M1, M5, M116, and M127 α-synuclein, are examined utilizing tandem mass spectrometry. Covalent labeling of proteins identifies solvent accessible residues and reveals the hydrophobicity (or hydrophilicity) of their microenvironment, while cross-linking of proteins maps the proximity of residue pairs. This work investigates how biologically relevant post-translational modifications alter the structural ensembles of alpha synuclein proteoforms. The combination of covalent labeling and cross-linking unequivocally demonstrates that phosphorylation at S129 stabilizes a more compact alpha synuclein conformation.