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

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 averagi...

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Bibliographic Details
Published in:Angewandte Chemie International Edition 2023-04, Vol.62 (15), p.e202216771-n/a
Main Authors: Chappard, Alexandre, Leighton, Craig, Saleeb, Rebecca S., Jeacock, Kiani, Ball, Sarah R., Morris, Katie, Kantelberg, Owen, Lee, Ji‐Eun, Zacco, Elsa, Pastore, Annalisa, Sunde, Margaret, Clarke, David J., Downey, Patrick, Kunath, Tilo, Horrocks, Mathew H.
Format: Article
Language:English
Subjects:
Aggregates
Aggregation or Oligomerization
alpha-Synuclein - chemistry
Amyloid
Amyloid - chemistry
Amyloidogenic Proteins
Antibodies
Chemical compounds
Confocal microscopy
Fluorescence
Fluorophores
Humans
Microfluidics
Microscopy
Movement disorders
Neurodegenerative diseases
Parkinson Disease - metabolism
Parkinson's disease
Protein Aggregates
Protein folding
Proteins
Single-Molecule
Synuclein
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Summary: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. Pathological protein aggregates in neurodegenerative disorders are difficult to characterise using current methods. We present a novel single‐molecule detection method to specifically detect and characterise α‐synuclein aggregates at picomolar concentrations. We demonstrate the ability to detect aggregates in biologically relevant samples.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.202216771