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.

Background: The 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 alpha-synuclein (aSYN) 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 aSYN quantification in human biological fluids (from Euroimmun, MSD, and Biolegend) are capable of capturing the diversity and complexity of relevant aSYN proteoforms. Methods: We investigated and compared the ability of the different assays to detect the diversity of aSYN proteoform using a library of aSYN proteins that compromise the majority of disease-relevant aSYN variants and post-translational modification. Results: Our findings showed that none of the three tested immunoassays accurately capture the totality of relevant aSYN species and are unable to recognize most disease-associated C-terminally truncated variants of aSYN. Moreover, several N-terminal truncations and phosphorylation/nitration differentially modify the level of aSYN detection and recovery by different immunoassays, and a CSF matrix effect was observed for most of the aSYN proteoforms analyzed by the three immunoassays. Conclusions: Our results showed that these immunoassays do not capture the totality of the relevant aSYN species and therefore may not be appropriate tools to provide an accurate measure of total aSYN levels in samples containing modified forms of the protein. This highlights the need for next-generation aSYN immunoassays that capture the diversity of aSYN proteoforms.