Two-color coincidence single-molecule pulldown for the specific detection of disease-associated protein aggregates.
Two-color coincidence single-molecule pulldown for the specific detection of disease-associated protein aggregates.
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DOI：
10.1126/sciadv.adi7359
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发表时间：
2023-11-17
期刊：
影响因子：
13.6
通讯作者：
Horrocks, Mathew H.
中科院分区：
文献类型：
--
作者：
Saleeb, Rebecca S.;Leighton, Craig;Lee, Ji-Eun;O'Shaughnessy, Judi;Jeacock, Kiani;Chappard, Alexandre;Cumberland, Robyn;Zhao, Tianxiao;Ball, Sarah R.;Sunde, Margaret;Clarke, David J.;Piche, Kristin;McPhail, Jacob A.;Louwrier, Ariel;Angers, Rachel;Gandhi, Sonia;Downey, Patrick;Kunath, Tilo;Horrocks, Mathew H.
蛋白质错误折叠和聚集是许多神经退行性疾病的特征，包括阿尔茨海默病和帕金森病。聚集过程中产生的寡聚体可能参与疾病的发病机制，并提出了有前途的生物标志物候选者。然而，由于其体积小，浓度低，仍然缺乏具体的工具来量化和表征复杂的生物样品中的聚集体。在这里，我们提出了单分子双色聚集下拉（STAPull），它克服了这一挑战，通过探测固定化蛋白质使用正交标记的检测抗体。通过分析共定位信号，我们可以消除单体蛋白质，并具体量化聚集蛋白质。使用聚集倾向的α-突触核蛋白蛋白作为模型，我们证明了这种方法可以特异性地检测聚集体，检测限为5皮摩尔。此外，我们表明STAPull可用于一系列样品，包括人类生物流体。STAPull适用于各种疾病的蛋白质聚集体，并将有助于鉴定在诊断这些疾病方面至关重要的生物标志物。提出了一种灵敏的检测方法，可以检测神经退行性疾病症状发作前的蛋白质结构。
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. A sensitive assay is presented that can detect the protein structures that precede symptom onset in neurodegenerative disease.
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DOI：
10.1016/j.bbapap.2010.04.001
发表时间：
2010-07
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Biancalana M;Koide S
通讯作者：
Koide S
影响因子：
15.1
作者：
Lasagna-Reeves CA;Castillo-Carranza DL;Sengupta U;Clos AL;Jackson GR;Kayed R
通讯作者：
Kayed R
影响因子：
16.6
作者：
通讯作者：
--
影响因子：
15.1
作者：
Majbour NK;Vaikath NN;van Dijk KD;Ardah MT;Varghese S;Vesterager LB;Montezinho LP;Poole S;Safieh-Garabedian B;Tokuda T;Teunissen CE;Berendse HW;van de Berg WD;El-Agnaf OM
通讯作者：
El-Agnaf OM
影响因子：
6.1
作者：
Kumar, Senthil T.;Jagannath, Somanath;Lashuel, Hilal A.
通讯作者：
Lashuel, Hilal A.