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Chan Hyun Na
Johns Hopkins University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-3622-2938
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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 (TiO2) 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.
Keywords
Parkinson’s disease, α-synuclein, phosphotyrosine, cerebrospinal fluid, parallel reaction monitoring
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CURRENT STATUS: Published
View the published article at Clinical Proteomics.
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Editorial decision: Accept
On 16 Apr, 2020
Editor assigned
On 09 Apr, 2020
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On 08 Apr, 2020
Editor invited
On 08 Apr, 2020
No comments provided
Editorial decision: Minor revision
On 11 Mar, 2020
Review #1 received
Received 04 Mar, 2020
Reviewer #3 agreed
On 03 Mar, 2020
Review #3 received
Received 03 Mar, 2020
Review #2 received
Received 03 Mar, 2020
Reviewers invited
Invitations sent on 28 Feb, 2020
Reviewer #1 agreed
On 28 Feb, 2020
Reviewer #2 agreed
On 28 Feb, 2020
Editor assigned
On 28 Feb, 2020
Submission checks complete
On 27 Feb, 2020
Editor invited
On 27 Feb, 2020
Version 1
Posted 06 Jan, 2020
No comments provided
Editorial decision: Major revision
On 28 Jan, 2020
Review #1 received
Received 19 Jan, 2020
Editor assigned
On 02 Jan, 2020
Reviewers invited
Invitations sent on 02 Jan, 2020
Reviewer #1 agreed
On 02 Jan, 2020
Reviewer #2 agreed
On 02 Jan, 2020
First submitted
On 01 Jan, 2020
Submission checks complete
On 01 Jan, 2020
Editor invited
On 01 Jan, 2020
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See the published version of this preprint at Clinical Proteomics.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Chan Hyun Na
Johns Hopkins University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-3622-2938
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Clinical Proteomics.
No community comments so far
Editorial decision: Accept
On 16 Apr, 2020
Editor assigned
On 09 Apr, 2020
Submission checks complete
On 08 Apr, 2020
Editor invited
On 08 Apr, 2020
No comments provided
Editorial decision: Minor revision
On 11 Mar, 2020
Review #1 received
Received 04 Mar, 2020
Reviewer #3 agreed
On 03 Mar, 2020
Review #3 received
Received 03 Mar, 2020
Review #2 received
Received 03 Mar, 2020
Reviewers invited
Invitations sent on 28 Feb, 2020
Reviewer #1 agreed
On 28 Feb, 2020
Reviewer #2 agreed
On 28 Feb, 2020
Editor assigned
On 28 Feb, 2020
Submission checks complete
On 27 Feb, 2020
Editor invited
On 27 Feb, 2020
Version 1
Posted 06 Jan, 2020
No comments provided
Editorial decision: Major revision
On 28 Jan, 2020
Review #1 received
Received 19 Jan, 2020
Editor assigned
On 02 Jan, 2020
Reviewers invited
Invitations sent on 02 Jan, 2020
Reviewer #1 agreed
On 02 Jan, 2020
Reviewer #2 agreed
On 02 Jan, 2020
First submitted
On 01 Jan, 2020
Submission checks complete
On 01 Jan, 2020
Editor invited
On 01 Jan, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Clinical Proteomics.
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 (TiO2) 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.
Figure 1
Figure 2
Figure 3
Figure 4
This is a list of supplementary files associated with this preprint. Click to download.
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