The aim of our study was to investigate cerebrospinal fluid (CSF) tryptic peptide profiles as potential diagnostic biomarkers for the discrimination of parkinsonian disorders. CSF samples were collected from individuals with parkinsonism, who had an uncertain diagnosis at the time of inclusion and who were followed for up to 12 years in a longitudinal study. We performed shotgun proteomics to identify tryptic peptides in CSF of Parkinson’s disease (PD, n=10), multiple system atrophy patients (MSA, n=5) and non-neurological controls (n=10). We validated tryptic peptides with differential levels between PD and MSA using a newly developed selected reaction monitoring (SRM) assay in CSF of PD (n=46), atypical parkinsonism patients (AP; MSA, n=17; Progressive supranuclear palsy; n=8) and non-neurological controls (n=39). We identified 191 tryptic peptides that differed significantly between PD and MSA, of which 34 met our criteria for SRM development. For 14/34 peptides we confirmed differences between PD and AP. These tryptic peptides discriminated PD from AP with moderate-to-high accuracy. Random forest modelling including tryptic peptides plus either clinical assessments or other CSF parameters (neurofilament light chain, phosphorylated tau protein) and age improved the discrimination of PD vs. AP. Our results show that discovery of tryptic peptides by untargeted and subsequent validation by targeted proteomics is a suitable strategy to identify novel potential CSF biomarkers for PD versus AP. Furthermore, the tryptic peptides, and corresponding proteins, that we identified as differential biomarkers may increase our current knowledge about the disease-specific pathophysiological mechanisms of parkinsonism.

The CSF samples for SRM and MS analysis were processed in randomized order using 50 µL of CSF from each patient as input. Prior to protein digestion, samples were subjected to overnight freeze-drying to concentrate the sample. On the next day, the sample was reconstituted with 4.3 µL of 8 M urea solution in 10 mM Tris-HCL, and diluted with 4.3 µL 10 mM Tris-HCL to reach a final urea concentration of 4 M. Protein reduction was performed by incubation with 0.5 µL of 10 mM dithiothreitol for 30 min at room temperature, followed by alkylation by incubation with 0.5 µL of 50 mM 2-chloroacetamide for 30 min at room temperature, kept in the dark. Samples were then incubated with 1 µL of 0.5 μg/μl Lysyl endopeptidase C for 3 h at room temperature, resulting in a volume of 10.6 µL, which was subsequently diluted four times with 31.6 µL of 50 mM ammonium bicarbonate and incubated with 1 µL of trypsin (1 μg trypsin / 50 μg protein) for 4 h at 37 °C. Digestion reaction was stopped by adding 4.8 µL of 10% trifluoroacetic acid. A cocktail of synthesized isotope-labelled “heavy” peptides (JPT, Germany) on the C-termini of the target peptides at either a lysine (13C615N2) or arginine (13C615N4) residue was added to each sample to allow peptide identification and relative quantification. Samples were cleaned by passing them over a 0.22 µm filter and stored at -80 oC until MS analysis. Samples (2 µL) were subjected to LC-MS analysis in randomized order on the Acquity MClass UPLC Xevo TQ-S (Waters), coupled with an ionKey / MS system using a Waters peptide BEH C18, 130 Å, 1.7 μm, 150 μmx100mm ionKey column for chromatographic separation using a 30 min linear gradient of acetonitrile ranging from 3 to 35% with 0.1% formic acid at a flow rate of 2 μl/min.

Cerebrospinal fluid samples from participants with parkinsonian disorders and non-neurological controls were digested from 50 µL of CSF