Development of a novel method for the quantification of tyrosine 39 phosphorylated α- And β-synuclein in human cerebrospinal fluid

Chan Hyun Na, Gajanan Sathe, Liana S. Rosenthal, Abhay R. Moghekar, Valina L. Dawson, Ted M. Dawson, Akhilesh Pandey

Research output: Contribution to journalArticle

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 (Y39) of α-synuclein (α-syn) (pY39 α-syn), which contributes to the death of dopaminergic neurons. Because pY39 α-syn may be pathogenic, monitoring pY39 α-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 α-syn can be detected in the cerebrospinal fluid (CSF) of PD patients by targeted mass spectrometry. Methods: Here, we report a two-step enrichment method in which phosphotyrosine peptides were first enriched with an anti-phosphotyrosine antibody followed by a second round of enrichment by titanium dioxide (TiO2) beads to detect EGVLpYVGSK sequence derived from tyrosine 39 region of α- and β-synuclein (αβ-syn). Accurate quantification was achieved by adding a synthetic heavy version of pY39 αβ-syn peptide before enzymatic digestion. Results: Using the developed enrichment methods and optimized parallel reaction monitoring (PRM) assays, we detected pY39 αβ-syn peptide in human CSF and demonstrated that the ratio of pY39 αβ-syn to Y39 αβ-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.

Original languageEnglish (US)
Article number13
JournalClinical Proteomics
Volume17
Issue number1
DOIs
StatePublished - May 4 2020

Keywords

  • Cerebrospinal fluid
  • Parallel reaction monitoring
  • Parkinson's disease
  • Phosphotyrosine
  • α-Synuclein
  • β-Synuclein

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Clinical Biochemistry

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