Abstract

Background: Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder. Biomarkers that can help monitor the progression of PD or response to disease-modifying agents will be invaluable in making appropriate therapeutic decisions. Further, biomarkers that could be used to distinguish PD from other related disorders with PD-like symptoms will be useful for accurate diagnosis and treatment. C-Abl tyrosine kinase is activated in PD resulting in increased phosphorylation of the tyrosine residue at position 39 of a-synuclein (a-syn) (pY39 a-syn), which contributes to the death of dopaminergic neurons. Because pY39a-syn may be pathogenic, monitoring pY39 a-syn could allow us to diagnose presymptomatic PD and help monitor disease progression and response to treatment. We sought to investigate if increased phosphorylation of pY39 a-syn can be detected in the CSF of PD patients by targeted mass spectrometry. Methods: Here, we report a two-step enrichment method in which phosphotyrosine peptides including pY39 a-syn containing peptide were first enriched with an anti-phosphotyrosine antibody followed by a second round of enrichment by titanium oxide (TiO 2 ) beads. Accurate quantification was achieved by the addition of a synthetic heavy version of pY39 a-syn peptide added before enzymatic digestion. Results: Using the developed enrichment methods and optimized parallel reaction monitoring assays, we detected pY39 a-syn peptide in human CSF and demonstrate that the ratio of pY39 a-syn to total a-syn was significantly increased in the CSF of patients with PD. Conclusions: We anticipate that this optimized two-step enrichment-based PRM detection method will help monitor c-Abl activation in PD patients and can also be used to quantify other phosphotyrosine peptides of low abundance in biological samples.