In the ever-evolving landscape of molecular biology, a transformative leap appears imminent: the sequencing of proteins at the single-molecule level via nanopore technology. This breakthrough, rigorously explored in recent research, signals a paradigm shift from DNA and RNA sequencing to the direct, real-time reading of polypeptides. For over thirty years, biological nanopore sequencing has revolutionized genomics by enabling the interrogation of nucleic acids in unprecedented detail. Now, pioneering work is pointing toward a new frontier — harnessing nanopores to decode the complex language of proteins with single-amino acid resolution.
The promise of protein nanopore sequencing is enormous. Unlike nucleic acids, proteins exhibit a far greater diversity arising from 20 standard amino acids and a vast spectrum of post-translational modifications (PTMs). This biochemical complexity has long stymied efforts for straightforward sequencing techniques. Traditional mass spectrometry methods, while powerful, struggle with sensitivity, quantification, and the detection of subtle modifications at the single-molecule scale. Nanopore-based protein sequencing could overcome these barriers by threading individual proteins through nanoscale pores and detecting sequential amino acid signals, thus enabling comprehensive readouts of proteomic heterogeneity in real time.
At the heart of this approach lies the exquisite electrical sensitivity of nanopores. When a protein or peptide molecule traverses a nanopore embedded in a membrane, it modulates an ionic current passing through the pore. These current disruptions carry rich information correlating to the biochemical identity of the molecule’s sequence. Over the past three decades, this principle has been successfully applied to sequence DNA and RNA by differentiating the nucleotides based on their distinct electrical signatures. Translating these methods to proteins presents unique technical challenges due to the greater chemical variety and the more complex physicochemical interplay between amino acids and the nanopore environment.
Recent studies outline several innovative strategies to tackle these challenges. One approach involves engineering the nanopores themselves to optimize sensitivity and selectivity toward protein sequences. By modifying pore geometry and surface chemistry, researchers aim to amplify the electrical signals corresponding to each amino acid and minimize noise. Another avenue focuses on controlling protein dynamics, using molecular motors or chemical tags to regulate the translocation speed, allowing more precise temporal resolution of the signals. Integrating these advances with sophisticated computational algorithms enhances the potential to accurately decode the current fluctuations into precise amino acid sequences.
A key advancement in this emerging field is the demonstration of single-amino acid discrimination. By examining the characteristic current blockade patterns generated as peptides translocate, experiments have resolved distinct profiles attributable to individual amino acids. This milestone suggests that, with optimized pore constructs and data analysis techniques, it will soon be feasible to read entire protein sequences at the level of individual residues. Such resolution dramatically surpasses current proteomics capabilities, opening avenues to directly identify sequence variants, rare isoforms, and subtle PTMs without the need for extensive sample preparation or population averaging.
The implications of nanopore protein sequencing extend deeply into biomedical research and clinical diagnostics. Proteins are the primary effectors of cellular function, and their diverse modifications regulate an astonishing breadth of biological processes. Being able to measure these molecular nuances at the single-molecule level will empower researchers to unravel complex disease mechanisms, discover novel biomarkers, and monitor therapeutic responses with unparalleled accuracy. For instance, detecting low-abundance proteins or rare pathological variants in complex biological fluids could revolutionize early diagnosis of diseases like cancer and neurodegeneration.
Furthermore, nanopore-based protein sequencing could transform the study of proteoforms — the diverse functional variants of proteins generated by alternative splicing, PTMs, and genetic polymorphisms. Mass spectrometry, while comprehensive, often loses the linkage information between modifications on the same protein molecule. Nanopores hold the promise of maintaining this linkage by sequencing intact, full-length proteins individually. This ability could illuminate proteoform diversity and functional heterogeneity that are currently obscured by ensemble averaging techniques.
Achieving practical protein nanopore sequencing devices necessitates overcoming challenges such as reproducible pore fabrication, protein capture efficiency, and data interpretation complexity. Precise control of protein unfolding and translocation speed is imperative to achieve high fidelity sequencing. Advances in protein engineering, such as site-specific labeling or motor proteins that ratchet the polypeptide through the pore in steady increments, are under intense investigation to address these demands. Concurrent development of machine learning models to interpret the vast and complex current signal datasets is equally vital.
Intriguingly, the modular nature of nanopore platforms offers potential synergy with other biotechnological tools. By integrating nanopore sequencing with microfluidics and single-cell proteomics workflows, researchers envision high-throughput, multiplexed analyses that could decode the protein composition of individual cells or subcellular compartments. Such capability would provide unprecedented insight into cellular heterogeneity and microenvironmental influences, revolutionizing fields from developmental biology to precision medicine.
The path towards routine single-molecule protein sequencing will likely involve iterative refinement bridging biophysics, engineering, computational biology, and biochemistry. Collaborative efforts harnessing expertise across these disciplines are advancing prototype nanopore sensors capable of distinguishing all 20 amino acids under experimental conditions mimicking physiological environments. Parallel progress in real-time data acquisition hardware and cloud-based analysis pipelines is facilitating rapid feedback loops to optimize experimental parameters.
While still at an early stage compared to nucleic acid nanopore sequencing, the momentum behind protein nanopore technology is striking. Commercial entities and academic laboratories alike are investing in this nascent field, motivated by the enormous market potential and the transformative scientific promise. Early-generation devices may initially provide partial sequence reads or focus on specific protein classes, but these stepping stones pave the way for comprehensive, full-length protein characterization platforms.
Looking ahead, the convergence of nanopore protein sequencing with artificial intelligence-driven interpretation could usher in an era where clinicians and researchers obtain detailed protein sequence information rapidly and cost-effectively. This capability would complement genomics and transcriptomics, completing the molecular picture from DNA code to functional protein effectors. The anticipated single-molecule resolution, combined with chemical specificity, stands to redefine our understanding of biology at the molecular level.
Ultimately, the realization of nanopore-based protein sequencing holds the potential to catalyze breakthroughs across science and medicine. By enabling direct observation of protein sequence variants, post-translational modifications, and splicing events on individual molecules, this technology could transform biomarker discovery, disease monitoring, and personalized therapies. As these emerging tools mature, they promise to equip researchers with a molecular microscope capable of deciphering life’s complexity with unprecedented clarity and speed.
In conclusion, biological nanopore sequencing is poised to extend its revolutionary impact beyond nucleic acids into the proteomic realm. The technical hurdles, though formidable, are steadily being overcome through innovative pore design, controlled translocation mechanisms, and advanced computational analytics. The prospect that nanopores will soon sequence entire proteins at single-amino acid resolution signals a looming revolution in proteomics, enabling precision insights into protein heterogeneity, modifications, and function at the single-molecule scale. This breakthrough promises to redefine biomedical research and clinical diagnostics for decades to come.
Subject of Research: Single-molecule protein sequencing using nanopores and its potential to achieve single-amino acid resolution.
Article Title: Toward single-molecule protein sequencing using nanopores
Article References:
Lu, C., Bonini, A., Viel, J.H. et al. Toward single-molecule protein sequencing using nanopores. Nat Biotechnol 43, 312–322 (2025). https://doi.org/10.1038/s41587-025-02587-y
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