A candidate reference method for the quantification of α-synuclein in cerebrospinal fluid using an SI traceable primary calibrator and multiple reaction monitoring

Objectives α-synuclein aggregation is an indicator of neurodegenerative diseases such as Parkinson’s disease (PD) and recent advances have suggested that this protein could serve as a potential biomarker. It has been indicated that soluble and oligomeric α-synuclein in biological fluids could have diagnostic applications for PD. Clinical laboratories currently rely on antibody-based assays to detect α-synuclein. These assays have limited specificity, low sensitivity and poor inter-lab reproducibility, which prevents the validation of α-synuclein as a biomarkers. This study aims to fill the unmet need for the standardisation of clinical measurements for α-synuclein. Methods We report the first candidate reference method for α-synuclein, using an SI traceable primary calibrator for α-synuclein and isotope dilution mass spectrometry. The primary calibrator was traceably quantified utilising a combination of amino acid analysis and nuclear magnetic resonance. A targeted sample clean-up procedure involving a non-denaturing Lys-C digestion and solid-phase extraction allowed for the sensitive detection of multiple proteotypic α-synuclein peptides in cerebrospinal fluid (CSF) samples. Results The candidate reference method procedure showed linearity across three orders of magnitude, covering the physiological levels of α-synuclein in CSF (LOQ = 0.1 ng/g). The method was used to quantify a cohort of CSF samples and the measurements were correlated with immunoassay-based quantifications. Conclusions The SI traceable quantification of α-synuclein in complex biological matrices means that the role of this protein can be further elucidated in synucleinopathies. This candidate reference method would lead to the harmonisation of α-synuclein measurements, which may allow for development of high throughput clinical tests.