Comparative Analysis of Total Alpha-Synuclein (αSYN) Immunoassays Reveals That They Do Not Capture the Diversity of Modified αSYN Proteoforms

BACKGROUND: The development of therapeutics for Parkinson’s disease (PD) requires the establishment of biomarker assays to enable stratifying patients, monitoring disease progression, and assessing target engagement. Attempts to develop diagnostic assays based on detecting levels of the α-synuclein (αSYN) protein, a central player in the pathogenesis of PD, have yielded inconsistent results. OBJECTIVE: To determine whether the three commercial kits that have been extensively used for total αSYN quantification in human biological fluids (from Euroimmun, MSD, and Biolegend) are capable of capturing the diversity and complexity of relevant αSYN proteoforms. METHODS: We investigated and compared the ability of the different assays to detect the diversity of αSYN proteoforms using a library of αSYN proteins that comprise the majority of disease-relevant αSYN variants and post-translational modifications (PTMs). RESULTS: Our findings showed that none of the three tested immunoassays accurately capture the totality of relevant αSYN species, and that these assays are unable to recognize most disease-associated C-terminally truncated variants of αSYN. Moreover, several N-terminal truncations and phosphorylation/nitration PTMs differentially modify the level of αSYN detection and recovery by different immunoassays, and a CSF matrix effect was observed for most of the αSYN proteoforms analyzed by the three immunoassays. CONCLUSION: Our results show that the tested immunoassays do not capture the totality of the relevant αSYN species and therefore may not be appropriate tools to provide an accurate measure of total αSYN levels in samples containing modified forms of the protein. This highlights the need for next generation αSYN immunoassays that capture the diversity of αSYN proteoforms.