Multiple rereads of single proteins at single-amino acid resolution using nanopores.
Multiple rereads of single proteins at single-amino acid resolution using nanopores.
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DOI：
10.1126/science.abl4381
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发表时间：
2021-12-17
期刊：
影响因子：
--
通讯作者：
Dekker C
中科院分区：
文献类型：
--
作者：
Brinkerhoff H;Kang ASW;Liu J;Aksimentiev A;Dekker C
能够识别单个蛋白质的蛋白质组学工具对细胞生物学的研究和应用具有重要意义。在这里，我们展示了一个基于纳米孔的单分子肽读取器，对单个肽内的单氨基酸取代敏感。DNA解旋酶Hel308将DNA-肽偶联物拉过生物纳米孔MspA。通过纳米孔读取离子电流信号，可以在单次读取中识别单氨基酸取代。分子动力学模拟表明，这些信号是由尺寸排斥和孔隙结合引起的。我们还展示了“倒带”肽读取的能力，获得了同一分子的许多独立读取，在单个氨基酸变异识别中产生的错误率<10−6。这些概念验证实验为单分子蛋白质指纹和分析技术的发展奠定了良好的基础。本文提出了概念验证实验和模拟的纳米孔为基础的方法线性读取单个肽与单个氨基酸取代的敏感性。
A proteomics tool capable of identifying single proteins would be important for cell biology research and applications. Here, we demonstrate a nanopore-based single-molecule peptide reader sensitive to single-amino-acid substitutions within individual peptides. A DNA-peptide conjugate was pulled through the biological nanopore MspA by the DNA helicase Hel308. Reading the ion current signal through the nanopore enabled discrimination of single-amino-acid substitutions in single reads. Molecular dynamics simulations showed these signals to result from size exclusion and pore binding. We also demonstrate the capability to ‘rewind’ peptide reads, obtaining numerous independent reads of the same molecule, yielding an error rate <10−6 in single amino acid variant identification. These proof-of-concept experiments constitute a promising basis for the development of a single-molecule protein fingerprinting and analysis technology. This paper presents proof-of-concept experiments and simulations of a nanopore-based approach for linearly reading individual peptides with sensitivity to single amino acid substitutions.
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影响因子：
3.3
作者：
Klauda, Jeffery B.;Venable, Richard M.;Freites, J. Alfredo;O'Connor, Joseph W.;Tobias, Douglas J.;Mondragon-Ramirez, Carlos;Vorobyov, Igor;MacKerell, Alexander D., Jr.;Pastor, Richard W.
通讯作者：
Pastor, Richard W.
影响因子：
5.5
作者：
Hart, Katarina;Foloppe, Nicolas;Baker, Christopher M.;Denning, Elizabeth J.;Nilsson, Lennart;MacKerell, Alexander D., Jr.
通讯作者：
MacKerell, Alexander D., Jr.
影响因子：
3.4
作者：
Aksimentiev, A;Schulten, K
通讯作者：
Schulten, K
影响因子：
3.4
作者：
Gumbart, James;Khalili-Araghi, Fatemeh;Sotomayor, Marcos;Roux, Benoit
通讯作者：
Roux, Benoit
影响因子：
4.4
作者：
JORGENSEN, WL;CHANDRASEKHAR, J;KLEIN, ML
通讯作者：
KLEIN, ML