Simultaneous quantification of tau and α-synuclein in cerebrospinal fluid by high-resolution mass spectrometry for differentiation of Lewy Body Dementia from Alzheimer's Disease and controls

Tau and alpha-synuclein are central in several neurodegenerative diseases, including Alzheimer Disease (AD), Dementia with Lewy Bodies (DLB) and Parkinson Disease (PD). New analytical methods for precise quantification of cerebrospinal fluid (CSF) levels of both tau and alpha-synuclein are required to differentiate between dementias or monitor therapeutic responses. Notably, levels of total alpha-synuclein reported by ELISA are inconsistent among studies, impacted by antibody specificity or lack of standardization. Here, we report on the development and validation of a sensitive and robust mass spectrometry-based assay for the simultaneous quantification of tau and alpha-synuclein in CSF. The optimized workflow avoided any affinity reagents, and involved the combination of two enzymes, Glu-C and trypsin for optimal sequence coverage of alpha-synuclein acidic C-terminus. Up to 7 alpha-synuclein peptides were quantified, including the C-terminal peptide (132-140), resulting in a sequence coverage of 54% in CSF. The lower limits of quantification (LLOQ) ranged from 0.1 ng mL(-1) to 1 ng mL(-1) depending on the peptide. Regarding CSF tau, 4 peptides common to all isoforms were monitored, and LLOQ ranged from 0.5 ng mL(-1) to 0.75 ng mL(-1). The multiplex method was successfully applied to CSF samples from AD and DLB patients, two clinically overlapping neurodegenerative diseases. CSF alpha-synuclein levels were significantly lower in DLB patients compared to AD and controls. Moreover, tau and alpha-synuclein concentrations showed opposite trends in AD and DLB patients, suggesting the benefit of combining the two biomarkers for differentiation of DLB from AD and controls.