Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates

Alexandre Chappard; Craig Leighton; Rebecca S. Saleeb; Kiani Jeacock; Sarah Ball; Katie Morris; Owen Kantelberg; Ji Eun Lee; Elsa Zacco; Annalisa Pastore; Margaret Sunde; David J. Clarke; Patrick Downey; Tilo Kunath; Mathew H. Horrocks

doi:10.1002/anie.202216771

Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates

Alexandre Chappard, Craig Leighton, Rebecca S. Saleeb, Kiani Jeacock, Sarah Ball, Katie Morris, Owen Kantelberg, Ji Eun Lee, Elsa Zacco, Annalisa Pastore, Margaret Sunde, David J. Clarke, Patrick Downey, Tilo Kunath, Mathew H. Horrocks^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract / Description of output

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.

Original language	English
Article number	e202216771
Journal	Angewandte Chemie - International Edition
Volume	62
Issue number	15
Early online date	10 Feb 2023
DOIs	https://doi.org/10.1002/anie.202216771
Publication status	Published - 3 Apr 2023

Keywords / Materials (for Non-textual outputs)

Aggregation or Oligomerization
Fluorescence
Microscopy
Proteins
Single-Molecule

Access to Document

10.1002/anie.202216771

20230210HorrocksAnieVoRFinal published version, 5.02 MBLicence: Creative Commons: Attribution (CC-BY)

https://onlinelibrary.wiley.com/doi/10.1002/anie.202216771

BBSRC EASTBIO Doctoral Training Partnership
Pennings, S.
1/09/18 → 1/09/22
Project: Other (Non-Funded/Miscellaneous)
Single-molecule detection of protein aggregates under flow
Horrocks, M. & Vendrell Escobar, M.
UK-based charities
18/07/18 → 17/07/19
Project: Research
Differentiation of GMP-grade human embryonic stem cells to midbrain dopaminergic neurons for transplantation
Kunath, T.
MRC
2/05/13 → 1/08/16
Project: Research

Cite this

Chappard, A., Leighton, C., Saleeb, R. S., Jeacock, K., Ball, S., Morris, K., Kantelberg, O., Lee, J. E., Zacco, E., Pastore, A., Sunde, M., Clarke, D. J., Downey, P., Kunath, T., & Horrocks, M. H. (2023). Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates. Angewandte Chemie - International Edition, 62(15), Article e202216771. https://doi.org/10.1002/anie.202216771

@article{501bb1d8d9594cf9b357904e9e318cee,

title = "Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates",

abstract = "Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.",

keywords = "Aggregation or Oligomerization, Fluorescence, Microscopy, Proteins, Single-Molecule",

author = "Alexandre Chappard and Craig Leighton and Saleeb, {Rebecca S.} and Kiani Jeacock and Sarah Ball and Katie Morris and Owen Kantelberg and Lee, {Ji Eun} and Elsa Zacco and Annalisa Pastore and Margaret Sunde and Clarke, {David J.} and Patrick Downey and Tilo Kunath and Horrocks, {Mathew H.}",

note = "Funding Information: We thank Kerry Tyson, Sarfaraj Topia and Hanna Hailu for reagent generation, and Ruodan Nan for Biacore data. The single‐molecule instruments used in this study were funded by the UK Dementia Research Institute, UCB Biopharmaceuticals, Alzheimer's Research UK (ARUK‐EG2018B‐004) and a kind donation from Dr. Jim Love. A.C. was supported by the Engineering and Physical Sciences Research Council and the MRC through the Centre for Doctoral Training in Optical Medical Imaging (grant no. EP/L016559/1), the Rosetrees Trust and the Scottish Funding Council (H14052/SIRL ID: 691). C.L. was supported by an MRC National Productivity Investment Fund studentship. R.S.S. and J.L. were supported by UCB Biopharma. R.S.S. was part‐funded by an EPSRC IAA grant and we would like to thank Prof. Andy Mount, Dean of Research at the College of Science and Engineering within the University of Edinburgh, and the EPSRC IAA support team at Edinburgh Innovations. K.J. was funded by the BBSRCEastBIO doctoral training program (BB/M010996/1). SRB was supported by a Research Training Program Scholarships from the Australian Government (Department of Education, Skills and Employment). O.K. was supported by a Scottish PhD Research & Innovation Network Traineeships in MND/MS. A.P. acknowledges funding from UK DRI (REI 3556) and Alzheimer's Research UK (ARUK‐PG2019B‐020). M.S. acknowledges funding from an Australian Research Council Discovery Project grant (DP200102463). A.P. was supported by Alzheimer's Research UK (ARUK‐PG2019B‐020). T.K. was supported by a MRC (MR/K017276/1) and Cure Parkinson's. Publisher Copyright: {\textcopyright} 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.",

year = "2023",

month = apr,

day = "3",

doi = "10.1002/anie.202216771",

language = "English",

volume = "62",

journal = "Angewandte Chemie - International Edition",

issn = "1433-7851",

publisher = "Wiley-VCH",

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}

Chappard, A, Leighton, C, Saleeb, RS, Jeacock, K, Ball, S, Morris, K, Kantelberg, O, Lee, JE, Zacco, E, Pastore, A, Sunde, M, Clarke, DJ, Downey, P, Kunath, T & Horrocks, MH 2023, 'Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates', Angewandte Chemie - International Edition, vol. 62, no. 15, e202216771. https://doi.org/10.1002/anie.202216771

TY - JOUR

T1 - Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates

AU - Chappard, Alexandre

AU - Leighton, Craig

AU - Saleeb, Rebecca S.

AU - Jeacock, Kiani

AU - Ball, Sarah

AU - Morris, Katie

AU - Kantelberg, Owen

AU - Lee, Ji Eun

AU - Zacco, Elsa

AU - Pastore, Annalisa

AU - Sunde, Margaret

AU - Clarke, David J.

AU - Downey, Patrick

AU - Kunath, Tilo

AU - Horrocks, Mathew H.

N1 - Funding Information: We thank Kerry Tyson, Sarfaraj Topia and Hanna Hailu for reagent generation, and Ruodan Nan for Biacore data. The single‐molecule instruments used in this study were funded by the UK Dementia Research Institute, UCB Biopharmaceuticals, Alzheimer's Research UK (ARUK‐EG2018B‐004) and a kind donation from Dr. Jim Love. A.C. was supported by the Engineering and Physical Sciences Research Council and the MRC through the Centre for Doctoral Training in Optical Medical Imaging (grant no. EP/L016559/1), the Rosetrees Trust and the Scottish Funding Council (H14052/SIRL ID: 691). C.L. was supported by an MRC National Productivity Investment Fund studentship. R.S.S. and J.L. were supported by UCB Biopharma. R.S.S. was part‐funded by an EPSRC IAA grant and we would like to thank Prof. Andy Mount, Dean of Research at the College of Science and Engineering within the University of Edinburgh, and the EPSRC IAA support team at Edinburgh Innovations. K.J. was funded by the BBSRCEastBIO doctoral training program (BB/M010996/1). SRB was supported by a Research Training Program Scholarships from the Australian Government (Department of Education, Skills and Employment). O.K. was supported by a Scottish PhD Research & Innovation Network Traineeships in MND/MS. A.P. acknowledges funding from UK DRI (REI 3556) and Alzheimer's Research UK (ARUK‐PG2019B‐020). M.S. acknowledges funding from an Australian Research Council Discovery Project grant (DP200102463). A.P. was supported by Alzheimer's Research UK (ARUK‐PG2019B‐020). T.K. was supported by a MRC (MR/K017276/1) and Cure Parkinson's. Publisher Copyright: © 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.

PY - 2023/4/3

Y1 - 2023/4/3

N2 - Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.

AB - Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.

KW - Aggregation or Oligomerization

KW - Fluorescence

KW - Microscopy

KW - Proteins

KW - Single-Molecule

U2 - 10.1002/anie.202216771

DO - 10.1002/anie.202216771

M3 - Article

AN - SCOPUS:85148941812

SN - 1433-7851

VL - 62

JO - Angewandte Chemie - International Edition

JF - Angewandte Chemie - International Edition

IS - 15

M1 - e202216771

ER -

Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates

Abstract / Description of output

Keywords / Materials (for Non-textual outputs)

Access to Document

Fingerprint

Projects

BBSRC EASTBIO Doctoral Training Partnership

Single-molecule detection of protein aggregates under flow

Differentiation of GMP-grade human embryonic stem cells to midbrain dopaminergic neurons for transplantation

Cite this