Cerebrospinal fluid proteomics implicates the granin family in Parkinson's disease

Abstract

Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Better understanding of the underlying disease mechanism(s) is an urgent need for the development of disease-modifying therapeutics. Limited studies have been performed in large patient cohorts to identify protein alterations in cerebrospinal fluid (CSF), a proximal site to pathology. We set out to identify disease-relevant protein changes in CSF to gain insights into the etiology of Parkinson's disease and potentially assist in disease biomarker identification. In this study, we used liquid chromatography-tandem mass spectrometry in data-independent acquisition (DIA) mode to identify Parkinson's-relevant biomarkers in cerebrospinal fluid. We quantified 341 protein groups in two independent cohorts (n = 196) and a longitudinal cohort (n = 105 samples, representing 40 patients) consisting of Parkinson's disease and healthy control samples from three different sources. A first cohort of 53 Parkinson's disease and 72 control samples was analyzed, identifying 53 proteins with significant changes (p < 0.05) in Parkinson's disease relative to healthy control. We established a biomarker signature and multiple protein ratios that differentiate Parkinson's disease from healthy controls and validated these results in an independent cohort. The second cohort included 28 Parkinson's disease and 43 control samples. Independent analysis of these samples identified 41 proteins with significant changes. Evaluation of the overlapping changes between the two cohorts identified 13 proteins with consistent and significant changes (p < 0.05). Importantly, we found the extended granin family proteins as reduced in disease, suggesting a potential common mechanism for the biological reduction in monoamine neurotransmission in Parkinson's patients. Our study identifies several novel protein changes in Parkinson's disease cerebrospinal fluid that may be exploited for understanding etiology of disease and for biomarker development.

Figure 1

Large-scale mass spectrometry-based biomarker discovery. (a) DIA-MS approach overview. (b–e) Library generation and data acquisition workflow. To generate the library for processing samples by DIA-MS, a representative sample subset (b) and fractionated CSF pool (c) were processed and run in DDA-mode. (d) The gene ontology (go)-term bioprocess (left) classification (pantherdb.org)^, of the protein library consisting of 341 quantifiable protein groups. Proteins involved in pathways implicated in PD are indicated on the right. (e) CSF samples (see Table 1) were processed and analyzed by DIA-MS. A representative total ion chromatogram is shown on right. SPE = solid phase extraction, DDA = data-dependent acquisition.

Figure 2

Univariate and multivariate analyses identify a PD-specific biomarker signature. (a) The univariate analyses of Cohort 1. Proteins highlighted in green and labeled by gene name have an AUC > 0.65 (red line) and an odds ratio (OR) > 2 or <0.5 (blue lines). (b) Whisker plots of the protein abundance of the top 4 predictive proteins. Significant protein changes of PD relative to the HC group are shown (Cohort 1 = C1; Cohort 2 = C2). (c,d) ROC analysis of the top proteins and protein ratios identified by Cohort 1. (e) ROC analysis of the predictive biomarker signature, (2.7(ATRN) + 1.2(C1QC) + 1.7(APOD)-2.2(SCG2) + 1.5(FBLN1)). (f) The calculated value of the biomarker signature as applied to the longitudinal cohort. (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 3

Thirteen proteins significantly change in both Cohort 1 and Cohort 2 in PD relative to HC. (a,b) Volcano plots of all proteins analyzed by DIA-MS for Cohort 1 (a) and Cohort 2 (b). Significant proteins identified in both cohorts are highlighted in green and labeled by gene name. The red line indicates a p-value cutoff of 0.05. (c) A Venn diagram displaying number of proteins with significant changes in Cohort 1 and 2. (d) Average fold change (right) compared to HC or individual sample (left) fold change normalized to mean for the 13 proteins (Top13) that were found to change in both cohorts by DIA-MS analysis in PD.

Figure 4

Peptide quantification reveals region-specific reduction of the extended granin family in PD. Granin peptides were found to either decrease in PD in both cohorts (red), one cohort (light red), or neither cohort (gray). The C-terminal region of PCSK1N, SCG2, and VGF are decreased in both PD cohorts relative to HC. For SCG3, the N-terminal region is decreased in PD.