Comparative analysis of total alpha-synuclein (αSYN) immunoassays reveals that they do not capture the diversity of modified αSYN proteoforms

BackgroundThe development of therapeutics for Parkinsons 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 -synuclein (SYN) protein, a central player in the pathogenesis of PD, have yielded inconsistent results. ObjectiveTo determine whether the three commercial kits that have been extensively used for total SYN quantification in human biological fluids (from Euroimmun, MSD, and Biolegend) are capable of capturing the diversity and complexity of relevant SYN proteoforms. MethodsWe investigated and compared the ability of the different assays to detect the diversity of SYN proteoform using a library of SYN proteins that compromise the majority of disease-relevant SYN variants and post-translational modification. ResultsOur findings showed that none of the three tested immunoassays accurately capture the totality of relevant SYN species and are unable to recognize most disease-associated C-terminally truncated variants of SYN. Moreover, several N-terminal truncations and phosphorylation/nitration differentially modify the level of SYN detection and recovery by different immunoassays, and a CSF matrix effect was observed for most of the SYN proteoforms analyzed by the three immunoassays. ConclusionsOur results showed that these immunoassays do not capture the totality of the relevant SYN species and therefore may not be appropriate tools to provide an accurate measure of total SYN levels in samples containing modified forms of the protein. This highlights the need for next-generation SYN immunoassays that capture the diversity of SYN proteoforms.