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

Objectives-synuclein aggregation is an indicator of neurodegenerative diseases such as Parkinsons disease (PD) and recent advances have suggested that this protein could serve as a potential biomarker. It has been indicated that soluble and oligomeric -synuclein in biological fluids could have diagnostic applications for PD. Clinical laboratories currently rely on antibody-based assays to detect -synuclein. These assays have limited specificity, low sensitivity and poor inter-lab reproducibility, which prevents the validation of -synuclein as a biomarkers. This study aims to fill the unmet need for the standardisation of clinical measurements for -synuclein. MethodsWe report the first candidate reference method for -synuclein, using an SI traceable primary calibrator for -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 -synuclein peptides in cerebrospinal fluid (CSF) samples. ResultsThe candidate reference method procedure showed linearity across three orders of magnitude, covering the physiological levels of -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. ConclusionsThe SI traceable quantification of -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 -synuclein measurements, which may allow for development of high throughput clinical tests.