Plasma α-synuclein domain profiles across α-synucleinopathies

Abstract

The differential diagnosis of α-synucleinopathies, including Parkinson's disease, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), remains challenging due to overlapping clinical features and the absence of reliable biomarkers. We developed a targeted mass spectrometry assay to profile α-synuclein peptides in plasma from Parkinson's disease (n = 82), DLB (n = 32), MSA (n = 8) and controls (n = 21). We hypothesized that disease-specific truncations or post-translational modifications would alter levels of non-modified α-synuclein peptides across α-synucleinopathies. The assay quantified non-modified peptides derived from the N-terminus and non-amyloid component (NAC) domain, regions implicated in aggregate formation. Although peptide levels were consistent across disease groups, a distinct NAC domain pattern observed in MSA may reflect unique pathological processes. This study presents the first blood-based profiling of α-synuclein peptides in these disorders, offering a basis for further investigation into disease mechanisms. Refinement of the assay to include post-translational modifications could enhance understanding of α-synucleinopathies and support future biomarker development.

Keywords: Lewy bodies; Parkinson’s disease; mass spectrometry; multiple system atrophy; α-synuclein.

Graphical Abstract

Figure 1

Group-wise comparison of α-synuclein and corresponding peptide levels in clinical samples. Individual plasma peptide levels from the N-terminal and NAC domains of α-synuclein: (A) α-Syn 13–21; (B) α-Syn 24–32; (C) α-Syn 35–43; (D) α-Syn 61–80; (E) α-Syn 81–96; (F) α-Syn combined; and (G) ELISA-based quantification of α-synuclein. Data are shown for individuals with DLB (n = 31), MSA (n = 8), Parkinson’s disease (n = 82) and CTRL (n = 21). The analysis includes individual boxplots for each peptide, a combined boxplot with mean Z-scored peptides representing total α-synuclein and a boxplot of ELISA results for α-synuclein. Statistical comparisons were performed using rank-based ANCOVA with age and sex as covariates, followed by a post hoc test with Bonferroni correction. No statistically significant effects of group were found for any variable (all P ≥ 0.425; F range: 0.03–0.94), and all post hoc pairwise tests were non-significant (P ≥ 0.05). The boxplots display the median, interquartile range and 1.5 interquartile range as a horizontal line, box and whiskers, respectively. The half violin plots illustrate data distribution. Each dot represents an individual sample measurement. The label ‘ns’ indicates a P > 0.05. α-Syn 13–21, α-synuclein amino acid residues 13–21; α-Syn 24–32, α-synuclein amino acid residues 24–32; α-Syn 35–43, α-synuclein amino acid residues 35–43; α-Syn 61–80, α-synuclein amino acid residues 61–80; α-Syn 81–96, α-synuclein amino acid residues 81–96; α-Syn combined, combined Z-scored peptide values representing total α-synuclein; ANCOVA, analysis of covariance; CTRL, controls; DLB, dementia with Lewy bodies; MSA, multiple system atrophy; ns, non-significant; PD, Parkinson’s disease; ratio L/H, the ratio of the light peptide and it’s isotopically labelled heavy form.

Figure 2

Profiling plasma α-synuclein peptides in clinical samples. (A) This figure displays the median abundance of α-synuclein peptides in plasma in different disease groups, Parkinson’s disease, CTRL, DLB and MSA. Each peptide is ordered along the x-axis by their amino acid position. The median abundances are depicted as coloured points, with error bars representing the interquartile ranges. The disease groups are colour coded: CTRL (teal), DLB (red), MSA (purple) and Parkinson’s disease (yellow). Lines connect the median values for each disease group across the different peptides to facilitate comparison. (B) The distribution of α-synuclein peptide abundances across the same disease groups. Each panel represents a different disease group, and within each panel, the abundance distributions of various peptides are shown using ridgeline plots. The peptides are ordered along the y-axis by their amino acid position. The ridgeline plots display the density of peptide abundances, with colours indicating the abundance levels.The x-axis represents the abundance levels, and the y-axis lists the peptides. No statistical tests were conducted for this figure. Median and interquartile ranges are shown to summarize the distribution of peptide abundance across disease groups. Abundance is shown as the ratio of light to heavy peptides and is therefore unitless. α-Syn 13–21, α-synuclein amino acid residues 13–21; α-Syn 24–32, α-synuclein amino acid residues 24–32; α-Syn 35–43, α-synuclein amino acid residues 35–43; α-Syn 61–80, α-synuclein amino acid residues 61–80; α-Syn 81–96, α-synuclein amino acid residues 81–96; CTRL, controls; DLB, dementia with Lewy bodies; MSA, multiple system atrophy; PD, Parkinson’s disease.

Figure 3

Correlogram depicting α-synuclein peptide correlations in plasma within each diagnostic group. The diagnostic groups include (A) Parkinson’s disease, (B) CTRL, (C) DLB and (D) MSA. The correlation matrices, visualized as correlograms, are based on Spearman's rank correlation coefficients. Circle size is proportional to the absolute value of the correlation coefficient, with larger circles indicating stronger correlations. Circle colour indicates the direction of the correlation. Darker shades represent stronger correlations. Significance levels are indicated as follows: *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001. α-Syn 13–21, α-synuclein amino acid residues 13–21; α-Syn 24–32, α-synuclein amino acid residues 24–32; α-Syn 35–43, α-synuclein amino acid residues 35–43; α-Syn 61–80, α-synuclein amino acid residues 61–80; α-Syn 81–96, α-synuclein amino acid residues 81–96; CTRL, controls; DLB, dementia with Lewy bodies; MSA, multiple system atrophy; PD, Parkinson’s disease.

Figure 4

ROC curves for classification based on α-synuclein peptide combinations. ROC curves illustrate the diagnostic accuracy of individual peptides and peptide combinations derived from logistic regression models. The curves depict the ability to differentiate (B) Parkinson’s disease from dementia with Lewy bodies (DLB), (C) from multiple system atrophy (MSA) and (A) from control groups. Highlighted are the five peptides or peptide combinations with the highest AUC values, indicating the most significant diagnostic performance. α-Syn 13–21, α-synuclein amino acid residues 13–21; α-Syn 24–32, α-synuclein amino acid residues 24–32; α-Syn 35–43, α-synuclein amino acid residues 35–43; α-Syn 61–80, α-synuclein amino acid residues 61–80; α-Syn 81–96, α-synuclein amino acid residues 81–96; AUC, area under the curve; CTRL, controls; DLB, dementia with Lewy bodies; MSA, multiple system atrophy; PD, Parkinson’s disease.