An alpha-synuclein MRM assay with diagnostic potential for Parkinson's disease and monitoring disease progression

Abstract

Aim: The alpha-synuclein (α-syn) level in human cerebrospinal fluid (CSF), as measured by immunoassays, is promising as a Parkinson's disease (PD) biomarker. However, the levels of total α-syn are inconsistent among studies with large cohorts and different measurement platforms. Total α-syn level also does not correlate with disease severity or progression. Here, the authors developed a highly sensitive MRM method to measure absolute CSF α-syn peptide concentrations without prior enrichment or fractionation, aiming to discover new candidate biomarkers.

Results: Six peptides covering 73% of protein sequence were reliably identified, and two were consistently quantified in cross-sectional and longitudinal cohorts. Absolute concentration of α-syn in human CSF was determined to be 2.1 ng/mL. A unique α-syn peptide, TVEGAGSIAAATGFVK (81-96), displayed excellent correlation with previous immunoassay results in two independent PD cohorts (p < 0.001), correlated with disease severity, and its changes significantly tracked the disease progression longitudinally.

Conclusions: An MRM assay to quantify human CSF α-syn was developed and optimized. Sixty clinical samples from cross-sectional and longitudinal PD cohorts were analyzed with this approach. Although further larger scale validation is needed, the results suggest that α-syn peptide could serve as a promising biomarker in PD diagnosis and progression.

Keywords: Diagnosis; MRM; Parkinson's disease; Progression; alpha-synuclein (α-syn).

Figure 1

Scheme of the LC-MS-MRM experiment. a. Human CSF samples preparation and analysis scheme. Equal amount of heavy isotope (¹⁵N) labeled recombinant α-syn was spiked into 200uL of human CSF samples. The CSF samples were then precipitated with TCA, digested with trypsin and AspN sequentially, and purified with C18 spin columns. The digested samples were analyzed with MRM LC-MS/MS. b. Calibration samples preparation and analysis scheme. Heavy isotope labeled and non-labeled light recombinant α-syn proteins were digested separately in two Eppendorf tubes, together with a matrix background of BSA. The digestion and purification procedures are the same as the CSF samples. Then the light α-syn peptides were serial diluted to form a calibration curve, while the heavy α-syn peptides were spiked into the light samples with a constant concentration and served as a normalization factor.

Figure 2

MRM results of peptide TVEGAGSIAAATGFVK in the UDALL cohort. a. MRM results correlated significantly with total α-syn levels measured with Luminex immunoassay (p = 0.0003). b. MRM results showed a significant decrease of α-syn levels between PD and control samples (p = 0.0355). c. Previous Luminex results showed a significant decrease of α-syn levels between PD and control samples (p = 0.0063). d. MRM results showed a significant correlation with UPDRS motor scores (p = 0.0007), which indicated PD severity.

Figure 3

MRM results of peptide TVEGAGSIAAATGFVK in the DATATOP cohort. a. MRM results significantly correlated with the total α-syn levels measured with Luminex immunoassay (p < 0.0001). b. MRM results showed a strong correlation with the UPDRS motor scores of the baseline samples (p = 0.0344). c. Trends between the patients’ MRM results and their cognitive statuses were observed with the Symbol Digit Modalities Test (p = 0.0062 for baseline samples and p = 0.0253 for final). d. Faster decrease of the peptide concentration of TVEGAGSIAAATGFVK indicated a more aggressive PD progression (p = 0.0167).