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 proteoform using a library of αSYN proteins that compromise the majority of disease-relevant αSYN variants and post-translational modification. Results Our findings showed that none of the three tested immunoassays accurately capture the totality of relevant αSYN species and are unable to recognize most disease-associated C-terminally truncated variants of αSYN. Moreover, several N-terminal truncations and phosphorylation/nitration 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. Conclusions Our results showed that these 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. ### Competing Interest Statement Hilal Lashuel (HAL) has received funding from industry to support research on neurodegenerative diseases, including from Merck Serono, UCB, Idorsia and Abbvie. These companies had no specific role in the in the conceptualization and preparation of and decision to publish this work. HAL is also the Co-founder and Chief Scientific Officer of ND BioSciences SA, a company that develops diagnostics and treatments for neurodegenerative diseases based on platforms that reproduce the complexity and diversity of proteins implicated in neurodegenerative diseases and their pathologies. Mohamed Bilal Fares (MBF) is the Co-founder and Director of R&D of ND BioSciences SA. Lara Petricca (LP) is the Head of Biomarkers and Diagnostic Development of ND BioSciences SA. Erik Stoops is an employee and shareholder of ADx NeuroSciences. The other authors declare no competing interest