Extrinsic Amyloid-Binding Dyes for Detection of Individual Protein Aggregates in Solution
Extrinsic Amyloid-Binding Dyes for Detection of Individual Protein Aggregates in Solution
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DOI：
10.1021/acs.analchem.8b02226
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发表时间：
2018-09-04
影响因子：
7.4
通讯作者：
Klenerman, David
中科院分区：
文献类型：
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作者：
Taylor, Christopher G.;Meisl, Georg;Klenerman, David
蛋白质聚集是一种关键的分子特征，是一系列神经退行性疾病的基础，包括阿尔茨海默病和帕金森病。要了解蛋白质聚集的分子细节，它是至关重要的，能够表征的阵列的异质聚集体形成的聚集过程中。我们在这里提出了一种高通量的方法来检测单个蛋白质聚集体，在溶液中，从无标记的聚集反应，我们证明了与帕金森氏病，asynuclein相关的蛋白质的方法。该方法结合了单分子共聚焦显微镜与一系列淀粉样蛋白结合的外源性染料，包括硫磺素T和五聚体甲酰噻吩乙酸，我们表明，我们可以观察到在低皮摩尔浓度的聚集体。单个聚集体的检测使我们能够量化它们的数量。此外，我们表明，这种方法也使我们能够从结合到聚集体的外来染料的发射强度获得结构上的见解。通过在单分子水平上分析聚集群体的时间演化，我们估计聚集体的碎片化率，这是病理聚集体倍增的关键过程。我们还表明，该方法允许检测这些聚集体的生物样品。在溶液中检测单个蛋白质聚集体的能力开辟了一系列新的应用，包括利用这种方法的潜力，高通量筛选人类生物流体的疾病诊断和早期检测。
Protein aggregation is a key molecular feature underlying a wide array of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. To understand protein aggregation in molecular detail, it is crucial to be able to characterize the array of heterogeneous aggregates that are formed during the aggregation process. We present here a high-throughput method to detect single protein aggregates, in solution, from a label-free aggregation reaction, and we demonstrate the approach with the protein associated with Parkinson's disease, asynuclein. The method combines single-molecule confocal microscopy with a range of amyloid-binding extrinsic dyes, including thioflavin T and pentameric formylthiophene acetic acid, and we show that we can observe aggregates at low picomolar concentrations. The detection of individual aggregates allows us to quantify their numbers. Furthermore, we show that this approach also allows us to gain structural insights from the emission intensity of the extrinsic dyes that are bound to aggregates. By analyzing the time evolution of the aggregate populations on a single-molecule level, we then estimate the fragmentation rate of aggregates, a key process that underlies the multiplication of pathological aggregates. We additionally demonstrate that the method permits the detection of these aggregates in biological samples. The capability to detect individual protein aggregates in solution opens up a range of new applications, including exploiting the potential of this method for high throughput screening of human biofluids for disease diagnosis and early detection.