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

Zhang, L.; Iles-Toth, E.; Cryar, A.; Drinkwater, G.; Di Vagno, L.; Pons, M.-L.; Mateyka, J.; McCullough, B.; Achtar, E.; Clarkson, C.; Goeschel, L.; Koertvelyessy, P.; Mussell, C.; Hopley, C.; Floeel, A.; Hirtz, C.; Lehmann, S.; Quaglia, M.

doi:10.1101/2024.03.20.585804

bioRxiv · 10.1101/2024.03.20.585804

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

Zhang, L.·Iles-Toth, E.·Cryar, A.·Drinkwater, G.·Di Vagno, L.·Pons, M.-L.·Mateyka, J.·McCullough, B.·Achtar, E.·Clarkson, C.·Goeschel, L.·Koertvelyessy, P.·Mussell, C.·Hopley, C.·Floeel, A.·Hirtz, C.·Lehmann, S.·Quaglia, M.

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Abstract

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.

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Zhang, L., Iles-Toth, E., Cryar, A., Drinkwater, G., Di Vagno, L., Pons, M.-L., Mateyka, J., McCullough, B., Achtar, E., Clarkson, C., Goeschel, L., Koertvelyessy, P., Mussell, C., Hopley, C., Floeel, A., Hirtz, C., Lehmann, S., Quaglia, M.. 2024-03-23. A candidate reference method for the quantification of α-synuclein in cerebrospinal fluid using an SI traceable primary calibrator and multiple reaction monitoring. https://doi.org/10.1101/2024.03.20.585804

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