Objective: Differentiating MSA from PD is clinically challenging. We developed CSF assays to measure full length and major C‑terminally truncated α‑synuclein (αSyn) species for potential differential diagnosis.

Background: Distinct pathogenic α‑synuclein fibril conformations, with different seeding activity in MSA than PD, suggest disease‑specific αSyn assemblies. There is also evidence of different post translationally truncated a-synuclein forms in MSA brains in comparison to PD.

Method: We developed an immunocapture-LC‑MS workflow with multi‑protease digestion to distinguish full‑length and major C‑terminally truncated αSyn proteoforms in CSF by quantifying proteoform‑specific signature peptides. A custom antibody targeting the C-terminal tryptic peptide enabled immunocapture from CSF, reducing sample complexity prior to a second digestion step with Glu-C. Blood contamination was assessed in parallel using a hemoglobin‑specific peptide to control for erythrocyte‑derived αSyn. The assay was tested on CSF samples from healthy individuals (18-55 years old), patients with PD (40-80 years old, Hoehn and Yahr ≤3), and patients with MSA (40-75 years old, with possible or probable MSA-P/MSA-C).

Results: The combined multi‑protease immunocapture LC‑MS/MS workflow enabled robust quantification of full‑length αSyn1–140 and the truncated proteoforms αSyn1–119 and αSyn1–122 in human CSF. Limits of quantification were 5pM for intact αSyn1–140 and 3pM for the truncated proteoforms using 500μL CSF. Although αSyn1–121 was initially targeted, it was excluded from the final method due to its absence in CSF samples. The hemoglobin‑specific peptide assay demonstrated acceptable performance for estimating blood contamination, with no clear correlation observed between hemoglobin levels and αSyn concentrations. Assay validity was supported by a statistically significant increase in the ratio of truncated to total αSyn in CSF from MSA patients compared with PD patients and healthy controls (p<0.0001).

Conclusion: Elevated levels of truncated α‑synuclein in CSF of MSA patients compared with PD and healthy controls highlight the potential of this assay to improve differential diagnosis and accelerate accurate clinical identification of MSA.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/a-csf-c-terminal-%ce%b1-synuclein-assay-to-differentiate-multiple-system-atrophy-from-parkinsons-disease/