复旦大学人类表型组研究所黄刚小组在研究中取得进展。他们报道了利用α-溶血素纳米孔获得窄分布的单分子肽信号，实现对肽的敏感识别。这一研究成果发表在2025年3月10日出版的国际学术期刊《美国化学会杂志》上。

该研究团队证明，用电泳力捕获抗电渗透流的肽可以在α-溶血素纳米孔中引起更均匀的阻塞。通过在pH3.8下用2M KCl浸泡缓冲液，课题组获得了最均匀的肽信号，这可能与肽的形状、线性化和实际驻留位置有关。五种具有乙酰化和磷酸化的肽，包括同分异构体肽，可以很容易地相互分离。精氨酸的瓜氨酸化取代和另一肽序列的β-羟基丁基化修饰也在混合物中进行了区分。用该方法对不同组成的多肽进行分析时，会产生均匀的肽阻滞。他们的工作提出了一种有效的方法来优化纳米孔信号，以α-溶血素纳米孔为主题进行肽分析。

研究人员表示，生物纳米孔技术已成为一种有前途的工具，用于分析肽和翻译后修饰在单分子水平。然而，目前由于肽的部分分离和低通量，主要是由于纳米孔检测的肽信号不均匀，限制了其广泛的应用。缩窄肽信号分布对于提高纳米孔的传感能力至关重要，但仍是一个瓶颈。

附：英文原文

Title: Obtaining Narrow Distributions of Single-Molecule Peptide Signals Enables Sensitive Peptide Discrimination with α-Hemolysin Nanopores

Author: Xing Wei, Jiaqi Wen, Hao Wu, Zhibei Qu, Gang Huang

Issue&Volume: March 10, 2025

Abstract: Biological nanopore technology has emerged as a promising tool for analyzing peptides and post-translational modifications at the single-molecule level. However, a broader application is currently limited by the partial separation of peptides and low-throughput, mainly due to the nonuniform peptide signals detected by nanopores. Narrowing the peptide signal distribution is crucial for improving the nanopore’s sensing ability but remains a bottleneck. Here, we demonstrate that capturing peptides with electrophoretic force against electroosmotic flow can provoke more uniform blockades in α-hemolysin nanopores. By using buffers with 2 M KCl at pH 3.8, we obtain the most uniform peptide signals, which may be correlated to the shape, linearization, and actual dwelling position of peptides. Five peptides with acetylation and phosphorylation, including isomeric peptides, can be readily separated from each other. The citrullination replacement of arginine and the β-hydroxybutyrylation modification in another peptide sequence are also discriminated in a mixture. A series of peptides with different compositions induced uniform peptide blockades when they were analyzed with our method. Our work presents an efficient approach to optimize nanopore signals for peptide analysis using α-hemolysin nanopores.

DOI: 10.1021/jacs.4c15469

Source: https://pubs.acs.org/doi/abs/10.1021/jacs.4c15469