The word “protein” comes from the Greek word “proteios”, which means of primary importance. Indeed, protein molecules, underlying virtually every process within cells, play a vital role in nearly all functions in the human body. For over a century, the vast majority of efforts in protein science have been devoted to resolve the structures and functions of proteins and bridge the gap between them. It is no doubt that the proteomic study is critical. However, unlike the well‐studied genomic related research, the development of peptide and protein science is hindered, which is challenged by limited characterization approaches. Nanopore sensing has been developing since the early 1990s and achieved DNA sequencing recently. This technique permits label‐free single‐molecule sensing with high sensitivity and selectivity, exhibiting promising potential for protein discrimination and sequencing. With increasing attention attracted to nanopore sensing of proteins, in this special issue of Small Methods, research at the cutting edges of protein science based on nanopore techniques are surveyed. These studies represent the most recent development in single‐protein sensing via a variety of nanopore systems, and illustrate the promising potential of nanopores in single‐molecule protein sequencing.

The nanopore‐based deciphering of proteomes needs to overcome a wide variety of challenges because the mature proteins are constituted by various amino acid types following complicated post‐translation modifications. In this special issue, fantastic exploration has been carried out from the aspects of single‐post‐translation‐modification discrimination, promoting distinct charged protein detection and controlled nanopore selectivity in a complex sample.

For biological nanopores, Geng et al. designed a mutant MspA nanopore as a nano‐confined space to enhance the interactions between the nanopore and peptides, contributing to the monitoring of protease activity (1900892); Kawano et al. designed a DNA probe to promote the nanopore sensitivity in discriminating single‐point‐mutation and resolved the corrections between unzipping kinetics and Gibbs energy of hybridization. This approach indicated a strategy for recognizing target protein by well controlling DNA‐protein interactions (2000101); Ying et al. engineered a well‐designed aerolysin single‐biomolecule interface with ultra‐high charge sensitivity, which achieves the determination of multiple phosphorylation sites on a single tau peptide (2000014).

For solid‐state nanopores, Radenovic and co‐workers developed an approach to realize wafer‐scale fabrication of atomically thin (2D) nanopores with high efficiency (2000072); Mayer et al. coated solid‐state nanopore with a polymer to minimize non‐specific interactions between nanopore and proteins, enhancing an accurate estimation of the volumes and spheroidal shapes for the freely translocating proteins (2000177); Balme's group fabricated conical track‐etched nanopores to characterize amyloid protein digestion (1900703). To overcome the limitation of the excessively fast translocation of protein in single‐molecule sensing based on solid‐state nanopores, Si et al. demonstrated that the use of nonionic detergents could slow down the translocation speed of amino acids in MoS₂ nanopore from molecular modelling (1900822). Moreover, Chen et al. modulated the electroosmotic flow (EOF) inside a solid‐state nanopore to slow down the streptavidin traversing (1900893). As for further improving sensitivity, Xia et al. introduced HRP enzyme and AIEgens into solid‐state nanopores to achieve the detection of H₂O₂, which was further applied to in situ and noninvasive monitoring of the releasing of H₂O₂ from living cells (1900432); Edel's group developed an aptamer‐functionalized nanopore extended filed‐effect transistors (nexFET) with improved performance on protein sensitivity and capture efficiency (2000356). In addition, Pelta et al. and Luchian et al. reviewed the advances of nanopore technology in protein discrimination and sequencing, and concluded with the open challenges for nanopore‐based protein sequencing (1900595, 2000090).

The articles in this special issue represent persuasively that nanopore technology exhibits the unambiguous advantage of detecting multiple proteins without labelling, and provides new strategies to accurately discriminate proteins, measure enzyme activity, and investigate protein‐protein interactions. It is worth noting that nature uses protein‐protein interactions to achieve ultra‐high accuracy in recognition of single peptides or protein substrates within the elaborated confinement. This enlightens us to employ protein‐protein interactions inside nanopore confinement to identify, and even sequence proteins in a real‐life sample (e.g., serum, tissue fluid and cytoplasm). After the rational design of the sensing interface and confined environment, the sensing performance of nanopores would be effectively promoted in sensitivity and selectivity. Moreover, the diameter‐controllable feature of solid‐state nanopores could extend the diversity of measurements from small biomolecules to large enzymes and antibodies. The papers in this special issue are only the tip of the iceberg of what is on the horizon in the coming years with new nanopore‐based protein science. Beyond fundamental science, owing to the remarkable features of nanopores with controllable sensitivity and selectivity, we envision that the nanopore‐based measurement strategy will find success in single‐molecule protein sequencing in the near future.

Finally, I would like to acknowledge all the authors’ excellent contributions and all the reviewers’ significant comments and assistance. Also sincere thanks to Editor‐in‐Chief Guang‐Chen Xu and editors Xi Wen and Lucie Kalvodova for their kind guidance and support.

“蛋白质”一词来自希腊语“ proteios””，这是最重要的。实际上，构成细胞内几乎每个过程的蛋白质分子在人体几乎所有功能中都起着至关重要的作用。一个多世纪以来，蛋白质科学领域的绝大多数工作都致力于解决蛋白质的结构和功能并弥合蛋白质之间的差距。毫无疑问，蛋白质组学研究至关重要。但是，与经过深入研究的基因组相关研究不同，肽和蛋白质科学的发展受到阻碍，这受到有限的表征方法的挑战。自1990年代初以来，纳米孔传感技术一直在发展，最近又实现了DNA测序。这项技术可以实现无标记的单分子传感，具有很高的灵敏度和选择性，在蛋白质识别和测序方面具有广阔的前景。小方法，基于纳米孔技术的蛋白质科学前沿研究进行了调查。这些研究代表了通过各种纳米孔系统进行单蛋白传感的最新进展，并说明了纳米孔在单分子蛋白测序中的潜在潜力。

基于纳米孔的蛋白质组学解密需要克服各种各样的挑战，因为成熟的蛋白质由复杂的翻译后修饰后的各种氨基酸类型组成。在本期特刊中，从单翻译后修饰识别的角度进行了出色的探索，从而促进了复杂样品中带电蛋白质检测和纳米孔选择性的控制。

对于生物纳米孔，耿等。设计了一个突变的MspA纳米孔作为纳米封闭空间，以增强纳米孔与肽之间的相互作用，从而有助于监测蛋白酶活性（1900892）；Kawano等。设计了一种DNA探针，以提高区分单点突变的纳米孔的敏感性，并解决了解链动力学和吉布斯杂交能之间的校正问题。这种方法表明了通过很好地控制DNA与蛋白质相互作用来识别目标蛋白质的策略（2000101）；Ying等。设计了一种具有超高电荷敏感性的精心设计的溶血素单生物分子界面，可测定单个tau肽上的多个磷酸化位点（2000014）。

对于固态纳米孔，拉德诺维奇及其同事开发了一种方法，可以高效地实现原子级薄（2D）纳米孔的晶圆级制造（2000072）；Mayer等。用聚合物包被的固态纳米孔，以最大程度地减少纳米孔与蛋白质之间的非特异性相互作用，从而增强对自由易位蛋白质的体积和球体形状的准确估算（2000177）；巴尔梅（Balme）的小组制造了圆锥形轨迹蚀刻纳米孔，以表征淀粉样蛋白的消化过程（1900703）。为了克服基于固态纳米孔的单分子传感中蛋白质过快转运的局限性，Si等人。证明使用非离子型去污剂会减慢MoS ₂中氨基酸的转运速度分子建模的纳米孔（1900822）。而且，Chen等。调节固态纳米孔中的电渗流（EOF）以减慢链霉亲和素的穿越速度（1900893）。至于进一步提高灵敏度，夏等。将HRP酶和AIEgens引入固态纳米孔以实现H ₂ O ₂的检测，该方法进一步应用于原位和无创监测H ₂ O ₂的释放来自活细胞（1900432）；Edel的小组开发了一种适体功能化的纳米孔扩展场效应晶体管（nexFET），该晶体管在蛋白质敏感性和捕获效率方面都有改进的性能（2000356）。此外，佩尔塔等。和Luchian等。回顾了纳米孔技术在蛋白质识别和测序方面的进展，并得出了基于纳米孔的蛋白质测序面临的开放挑战（1900595、2000090）。

本期特刊中的文章很有说服力地表示，纳米孔技术展现出无需标记即可检测多种蛋白质的明确优势，并提供了准确区分蛋白质，测量酶活性和研究蛋白质与蛋白质相互作用的新策略。值得注意的是，自然界利用蛋白质-蛋白质相互作用来实现超高精度，从而在精细范围内识别单个肽或蛋白质底物。这启发我们利用纳米孔限制内的蛋白质-蛋白质相互作用来鉴定甚至测序真实生活样品（例如血清，组织液和细胞质）中的蛋白质。通过合理设计传感界面和密闭环境，可以有效提高纳米孔的传感性能。此外，固态纳米孔的直径可控特征可以将测量范围从小型生物分子扩展到大型酶和抗体。本期特刊的文章只是未来几年基于新的基于纳米孔的蛋白质科学发展的冰山一角。除了基础科学之外，由于纳米孔具有可控制的灵敏度和选择性的显着特征，我们预见基于纳米孔的测量策略将在不久的将来在单分子蛋白质测序中获得成功。本期特刊的文章只是未来几年基于新的基于纳米孔的蛋白质科学发展的冰山一角。除了基础科学之外，由于纳米孔具有可控制的灵敏度和选择性的显着特征，我们预见基于纳米孔的测量策略将在不久的将来在单分子蛋白质测序中获得成功。本期特刊的文章只是未来几年基于新的基于纳米孔的蛋白质科学发展的冰山一角。除了基础科学之外，由于纳米孔具有可控制的灵敏度和选择性的显着特征，我们预见基于纳米孔的测量策略将在不久的将来在单分子蛋白质测序中获得成功。

最后，我要感谢所有作者的杰出贡献以及所有审稿人的重要评论和帮助。衷心感谢徐光诚总编和编辑Xi Wen和Lucie Kalvodova的友好指导和支持。