B-258 Development and Fit-for-Purpose Analytical Validation of a LC-MS/MS Method for the Quantification of pS129 Alpha-synuclein in Human Serum

Abstract

Background Parkinson’s Disease (PD) poses significant challenges due to its long progression period and overlapping pathology with other neurodegenerative diseases. The phosphorylation of alpha-synuclein at serine 129 (pS129) has been shown to play a crucial role in PD progression, and potentially serve as a diagnostic biomarker for PD. Methods This assay development and validation was conducted in the Clinical Diagnostics Laboratory department of Eli Lilly & Company using an Orbitrap Mass Spectrometer. The assay method uses a pS129-specific antibody for immunoprecipitation (IP), followed by sample clean-up with solid-phase extraction (SPE) and protein digestion using trypsin (Waters, RapiZyme). After digestion, tryptic peptides were separated, and the alpha-synuclein (Asyn) peptide (aa81-96) was quantified with a Parallel Reaction Monitoring (PRM) method. A five-point calibration curve (30, 100, 300, 1000, 2000 pg/mL) was prepared by spiking in pS129 Asyn protein in 2% SigMatrix (Sigma-Aldrich). Low and high concentration quality control (QC) samples were run alongside the validation samples on each plate. A heavy-labeled tryptic internal standard peptide was added to each sample for normalization. Data analysis was conducted using Skyline Software (MacCoss lab, University of Washington). Assay precision, accuracy, sensitivity, parallelism, dilution linearity, interference, and sample stability were evaluated in this analytical validation. Results Precision across 65 measurements from five unique samples (3 healthy and 2 diseased) ranging from 200 pg/mL to 1500 pg/mL showed total CV ranging between 13% to 16%, for individual samples. Spike recovery (Accuracy) was evaluated by adding recombinant pS129 Asyn protein to both healthy and diseased samples at concentrations of 0 pg/mL (No spike), 200 pg/mL, and 500 pg/mL. The percentage recovery (%RE) range for accuracy was between 106% to 132%. No interference was observed from non-phosphorylated Asyn protein, hemoglobin, or intralipid. The limit of blank was 47 pg/mL. Functional analytical sensitivity (lower limit of quantitation) was at 81 pg/mL. The stability study conducted over a period of seven days at 4°C showed %RE ranging from 97% to 122%. Samples remained stable for up to five freeze/thaw cycles at -20°C and -80°C. The final analytical measurement range was 80-2000 pg/mL. The assay showed good parallelism (up to 1:4 dilution, %RE: 107%-112%) and dilutional linearity (up to 1:4 dilution, %RE: 73%-103%). Conclusion Analytical validation shows that the assay method is robust for detecting pS129 Asyn in human serum, which can be crucial for research and clinical applications related to neurodegenerative diseases.