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 alpha-synuclein (alpha SYN) 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 alpha SYN quantification in human biological fluids (from Euroimmun, MSD, and Biolegend) are capable of capturing the diversity and complexity of relevant alpha SYN proteoforms.

Methods: We investigated and compared the ability of the different assays to detect the diversity of alpha SYN proteoforms using a library of alpha SYN proteins that comprise the majority of disease-relevant alpha SYN variants and post-translational modifications (PTMs).

Results: Our findings showed that none of the three tested immunoassays accurately capture the totality of relevant alpha SYN species, and that these assays are unable to recognize most disease-associated C-terminally truncated variants of alpha SYN. Moreover, several N-terminal truncations and phosphorylationinitration PTMs differentially modify the level of alpha SYN detection and recovery by different immunoassays, and a CSF matrix effect was observed for most of the alpha SYN proteoforms analyzed by the three immunoassays.

Conclusion: Our results show that the tested immunoassays do not capture the totality of the relevant alpha SYN species and therefore may not be appropriate tools to provide an accurate measure of total alpha SYN levels in samples containing modified forms of the protein. This highlights the need for next generation alpha SYN immunoassays that capture the diversity of alpha SYN proteoforms.