Visualizing Protein Aggregates at the Single-Molecule Level

Protein misfolding and aggregation are key features observed in numerous neurodegenerative conditions, such as Alzheimer's and Parkinson's disease. The oligomers formed during the aggregation process likely play a significant role in the development of these diseases and represent promising candidates for biomarkers. Nevertheless, due to their small size and low concentrations, there is a current lack of specific tools for quantifying and characterizing these aggregates in complex biological samples. In this presentation, I will introduce our recently devised single-molecule techniques, designed to overcome this obstacle by investigating immobilized proteins using detection antibodies labeled in an orthogonal manner. Through the analysis of colocalized signals, we can eliminate monomeric protein, and accurately quantify aggregated proteins. Using the aggregation-prone alpha-synuclein protein as a model, we illustrate that this approach can selectively identify aggregates with a detection limit of 5 picomolar. Furthermore, we demonstrate its applicability across various samples, including human biofluids. Our method is versatile and can be applied to protein aggregates associated with diverse disorders, contributing to the identification of crucial biomarkers for improved diagnostic efforts in these diseases.

Learning Objectives: 

1. Review the relevance of protein aggregates in neurodegenerative disorders.

2. Summarize how they can be visualized specifically at the single-molecule level.

3. Explain the concepts of super-resolution microscopy, and how it can be utilized for protein aggregate detection.