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Title: An alpha-synuclein MRM assay with diagnostic potential for Parkinson's disease and monitoring disease progression

Abstract

Aim: The alpha-synuclein (α-syn) level in human cerebrospinal fluid (CSF), as measured by immunoassays, is promising as a Parkinson’s disease (PD) biomarker. However, the levels of total α-syn are inconsistent among studies with large cohorts and different measurement platforms. Total α-syn level also does not correlate with disease severity or progression. Here, we developed a highly sensitive Multiple Reaction Monitoring (MRM) method to measure absolute CSF α-syn peptide concentrations without prior enrichment or fractionation, aiming to discover new candidate biomarkers. Results: Six peptides covering 73% of protein sequence were reliably identified, and two were consistently quantified in cross-sectional and longitudinal cohorts. Absolute concentration of α-syn in human CSF was determined to be 2.1ng/mL. A unique α-syn peptide, TVEGAGSIAAATGFVK (81-96), displayed excellent correlation with previous immunoassay results in two independent PD cohorts (p < 0.001), correlated with disease severity, and its changes significantly tracked the disease progression longitudinally. Conclusions: An MRM assay to quantify human CSF α-syn was developed and optimized. Sixty clinical samples from cross-sectional and longitudinal PD cohorts were analyzed with this approach. Although further larger-scale validation is needed, the results suggest that α-syn peptide could serve as a promising biomarker in PD diagnosis and progression.

Authors:
 [1];  [1];  [1];  [1];  [1];  [2];  [1];  [1];  [1];  [1];  [3];  [3];  [4];  [5];  [6]; ORCiD logo [7]
  1. Department of Pathology, University of Washington, Seattle WA USA
  2. Department of Pathology, University of Washington, Seattle WA USA; Department of Pathology, No. 3 Hospital of Beijing University, Beijing China
  3. Pacific Northwest National Laboratory, Richland WA USA
  4. Parkinson's Disease Research and Geriatric Research, Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle WA USA; Department of Neurology, University of Washington School of Medicine, Seattle WA USA
  5. Department of Neurology, University of Washington School of Medicine, Seattle WA USA
  6. Department of Neurology, Oregon Health and Science University, Portland OR USA
  7. Department of Pathology, University of Washington, Seattle WA USA; Department of Pathology, Peking University Health Science Centre and Third Hospital, Beijing 100083 China
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1378039
Report Number(s):
PNNL-SA-124743
Journal ID: ISSN 1862-8346; WN9030198
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: PROTEOMICS - Clinical Applications; Journal Volume: 11; Journal Issue: 7-8
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; parkinson’s disease; SRM; a-syn; diagnosis; progression

Citation Formats

Yang, Li, Stewart, Tessandra, Shi, Min, Pottiez, Gwenael, Dator, Romel, Wu, Rui, Aro, Patrick, Schuster, Robert J., Ginghina, Carmen, Pan, Catherine, Gao, Yuqian, Qian, Weijun, Zabetian, Cyrus P., Hu, Shu-Ching, Quinn, Joseph F., and Zhang, Jing. An alpha-synuclein MRM assay with diagnostic potential for Parkinson's disease and monitoring disease progression. United States: N. p., 2017. Web. doi:10.1002/prca.201700045.
Yang, Li, Stewart, Tessandra, Shi, Min, Pottiez, Gwenael, Dator, Romel, Wu, Rui, Aro, Patrick, Schuster, Robert J., Ginghina, Carmen, Pan, Catherine, Gao, Yuqian, Qian, Weijun, Zabetian, Cyrus P., Hu, Shu-Ching, Quinn, Joseph F., & Zhang, Jing. An alpha-synuclein MRM assay with diagnostic potential for Parkinson's disease and monitoring disease progression. United States. doi:10.1002/prca.201700045.
Yang, Li, Stewart, Tessandra, Shi, Min, Pottiez, Gwenael, Dator, Romel, Wu, Rui, Aro, Patrick, Schuster, Robert J., Ginghina, Carmen, Pan, Catherine, Gao, Yuqian, Qian, Weijun, Zabetian, Cyrus P., Hu, Shu-Ching, Quinn, Joseph F., and Zhang, Jing. Wed . "An alpha-synuclein MRM assay with diagnostic potential for Parkinson's disease and monitoring disease progression". United States. doi:10.1002/prca.201700045.
@article{osti_1378039,
title = {An alpha-synuclein MRM assay with diagnostic potential for Parkinson's disease and monitoring disease progression},
author = {Yang, Li and Stewart, Tessandra and Shi, Min and Pottiez, Gwenael and Dator, Romel and Wu, Rui and Aro, Patrick and Schuster, Robert J. and Ginghina, Carmen and Pan, Catherine and Gao, Yuqian and Qian, Weijun and Zabetian, Cyrus P. and Hu, Shu-Ching and Quinn, Joseph F. and Zhang, Jing},
abstractNote = {Aim: The alpha-synuclein (α-syn) level in human cerebrospinal fluid (CSF), as measured by immunoassays, is promising as a Parkinson’s disease (PD) biomarker. However, the levels of total α-syn are inconsistent among studies with large cohorts and different measurement platforms. Total α-syn level also does not correlate with disease severity or progression. Here, we developed a highly sensitive Multiple Reaction Monitoring (MRM) method to measure absolute CSF α-syn peptide concentrations without prior enrichment or fractionation, aiming to discover new candidate biomarkers. Results: Six peptides covering 73% of protein sequence were reliably identified, and two were consistently quantified in cross-sectional and longitudinal cohorts. Absolute concentration of α-syn in human CSF was determined to be 2.1ng/mL. A unique α-syn peptide, TVEGAGSIAAATGFVK (81-96), displayed excellent correlation with previous immunoassay results in two independent PD cohorts (p < 0.001), correlated with disease severity, and its changes significantly tracked the disease progression longitudinally. Conclusions: An MRM assay to quantify human CSF α-syn was developed and optimized. Sixty clinical samples from cross-sectional and longitudinal PD cohorts were analyzed with this approach. Although further larger-scale validation is needed, the results suggest that α-syn peptide could serve as a promising biomarker in PD diagnosis and progression.},
doi = {10.1002/prca.201700045},
journal = {PROTEOMICS - Clinical Applications},
number = 7-8,
volume = 11,
place = {United States},
year = {Wed Apr 19 00:00:00 EDT 2017},
month = {Wed Apr 19 00:00:00 EDT 2017}
}
  • Alpha-Synuclein (AS) fibrils constitute the major proteinaceous component of Lewy bodies (LBs), the pathological hallmark of Parkinson’s disease (PD) and other neurodegenerative diseases. Three single point mutations in the AS gene, as well as multiplication of the wild-type (WT) AS allele, have been previously identified in families with early-onset PD. Although AS fibrils have been the subject of intense study, critical details about their structure including the precise location of the B-strands and the extent of the core, the three-dimensional structure and the effects of the mutations—remain unknown. Here, we have used magic-angle spinning solid-state NMR spectroscopy to present amore » detailed characterization of the full-length WT AS fibrils. With improved sample preparations, isotopic labeling patterns and NMR experiments, we have confidently assigned more than 90% of the 13C and 15N backbone and sidechain chemical shifts of the detected residues from residue 39 to 97, and quantified the conformational dynamics throughout this region. Our results demonstrate that the core of AS fibrils extends with a repeated motif and that residues 30, 46 and 53-the early-onset PD mutant sites-are located in structured regions of AS fibrils.« less
  • The aggregation of {alpha}-synuclein is a hallmark feature of Parkinson's disease (PD) and other synucleinopathies. Metals are the significant etiological factors in PD, and their interaction with {alpha}-synuclein affect dramatically the kinetics of fibrillation in vitro and are proposed to play an important and potential neurodegenerative role in vivo. In the present study, we investigated the stoichiometry of binding of copper [Cu (II)] and iron [Fe (III)] with {alpha}-synuclein (wild recombinant type and A30P, A53T, E46K mutant forms) using isothermal titration calorimetry (ITC). {alpha}-Synuclein monomer (wild and mutant forms) titrated by Cu (II), showed two binding sites, with an apparentmore » K {sub B} of 10{sup 5} M and 10{sup 4} M, respectively. But, {alpha}-synuclein (wild type and mutant forms) titrated with Fe (III) revealed a K {sub B} of 10{sup 5} M with single binding site. The present investigation uncovers the detailed binding propensities between metals and {alpha}-synuclein and has biological implications in PD.« less
  • Understanding {alpha}-synuclein in terms of fibrillization, aggregation, solubility and stability is fundamental in Parkinson's disease (PD). The three familial mutations, namely, A30P, E46K and A53T cause PD because the hydrophobic regions in {alpha}-synuclein acquire {beta}-sheet configuration, and have a propensity to fibrillize and form amyloids that cause cytotoxicity and neurodegeneration. On simulating the native form and mutants (A30P, E46K and A53T) of {alpha}-synuclein in water solvent, clear deviations are observed in comparison to the all-helical 1XQ8 PDB structure. We have identified two crucial residues, {sup 40}Val and {sup 74}Val, which play key roles in {beta}-sheet aggregation in the hydrophobic regionsmore » 36-41 and 68-78, respectively, leading to fibrillization and amyloidosis in familial (A53T) PD. We have also identified V40D{sub V}74D, a double mutant of A53T (the most amyloidogenic mutant). The simultaneous introduction of these two mutations in A53T nearly ends its aggregation propensity, increases its solubility and positively enhances its thermodynamic stability.« less