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

TY - JOUR

T1 - Development of a novel method for the quantification of tyrosine 39 phosphorylated α- And β-synuclein in human cerebrospinal fluid

AU - Na, Chan Hyun

AU - Sathe, Gajanan

AU - Rosenthal, Liana S.

AU - Moghekar, Abhay R.

AU - Dawson, Valina L.

AU - Dawson, Ted M.

AU - Pandey, Akhilesh

N1 - Publisher Copyright: © 2020 The Author(s).

PY - 2020/5/4

Y1 - 2020/5/4

N2 - 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.

AB - 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.

KW - Cerebrospinal fluid

KW - Parallel reaction monitoring

KW - Parkinson's disease

KW - Phosphotyrosine

KW - α-Synuclein

KW - β-Synuclein

UR - https://www.scopus.com/pages/publications/85084393276

U2 - 10.1186/s12014-020-09277-8

DO - 10.1186/s12014-020-09277-8

M3 - Article

C2 - 32390785

AN - SCOPUS:85084393276

SN - 1542-6416

VL - 17

JO - Clinical proteomics

JF - Clinical proteomics

IS - 1

M1 - 13

ER -