학술논문
Two-color coincidence single-molecule pulldown for the specific detection of disease-associated protein aggregates.
Document Type
Article
Author
Saleeb, Rebecca S.; Leighton, Craig; Ji-Eun Lee; O'Shaughnessy, Judi; Jeacock, Kiani; Chappard, Alexandre; Cumberland, Robyn; Tianxiao Zhao; Ball, Sarah R.; Sunde, Margaret; Clarke, David J.; Piché, Kristin; McPhail, Jacob A.; Louwrier, Ariel; Angers, Rachel; Gandhi, Sonia; Downey, Patrick; Kunath, Tilo; Horrocks, Mathew H.
Source
Subject
Language
ISSN
2375-2548
Abstract
Protein misfolding and aggregation is a characteristic of many neurodegenerative disorders, including Alzheimer's and Parkinson's disease. The oligomers generated during aggregation are likely involved in disease pathogenesis and present promising biomarker candidates. However, owing to their small size and low concentration, specific tools to quantify and characterize aggregates in complex biological samples are still lacking. Here, we present single-molecule two-color aggregate pulldown (STAPull), which overcomes this challenge by probing immobilized proteins using orthogonally labeled detection antibodies. By analyzing colocalized signals, we can eliminate monomeric protein and specifically quantify aggregated proteins. Using the aggregation-prone alpha-synuclein protein as a model, we demonstrate that this approach can specifically detect aggregates with a limit of detection of 5 picomolar. Furthermore, we show that STAPull can be used in a range of samples, including human biofluids. STAPull is applicable to protein aggregates from a variety of disorders and will aid in the identification of biomarkers that are crucial in the effort to diagnose these diseases. [ABSTRACT FROM AUTHOR]