Résumé
Background
Synucleins (alpha, beta, and gamma) are potential biomarkers for Parkinson’s disease dementia (PDD), Dementia of Lewy body (DLB) and the Multi system atrophy (MSA). Their quantification with Mass spectrometry (MS) represents an interesting approach as it can precisely quantify synuclein proteoforms including mutations, alternative splicing, phosphorylation and other post‐translational modifications.
Methods
We developed an antibody free method starting with 95 µl of plasma. Patient cohort of 155 samples was analyzed with 57% PD, 22% LBD, 6% MSA, and 15% of control (CTRL) patient samples. Briefly, plasma proteins were differentially precipitated, clean‐up on AssayMap BRAVO (Agilent Technologies) and digest with trypsin/lys C. Liquid Chromatography‐Multiple Reaction Monitoring (LC‐MRM, Nexera Mikros – 8060, Shimadzu) method was developed to monitor 14 peptides originated from the alpha, beta and gamma synuclein sequence and one hemoglobin peptide. Method was validated following the EMA guidelines and compare to alpha synuclein immunodetection with Meso Scale Discovery platform (U‐plex human alpha synuclein kit).
Results
Quantitative assay based on LC‐MRM was validated on total alpha (140aa), beta (134aa) and gamma (127aa) synuclein peptides, and 4 alpha synuclein proteoforms (126, 112, 98 and 41) using 3 proteotypic peptides (one for 126/98, one for 112/98 and one for 41). After cohort quantification, alpha and beta synucleins were quantified in plasma with good performances and are able to statistically discriminate CTRL vs PD; CTRL vs LBD; CTRL vs MSA, and MSA vs PD. A slight correlation was found between the quantities obtain between MSD and MS assays.
Conclusion
MS based assay was successfully developed and validated to plasma synuclein proteoforms. Analysis of patient cohort indicate the ability of this assay to discriminate PD, DLB and MSA with a specificity of 81% and a sensitivity of 87%.