Breakthrough towards protein sequencing using Oxford Nanopore holds promise for disease research and drug development

New protein sequencing ‘proof-of-concept' method published in Nature shows promise in sequencing long, intact protein strands at single molecule resolution.

In a research collaboration with Oxford Nanopore Technologies, scientists from the University of Washington have unveiled a groundbreaking ‘proof-of-concept’ method showcasing the capability of the Oxford Nanopore sensing platform to read single protein molecules, a major step forward in protein analysis. In a study published today in Nature, the innovative technique enables researchers to read long, intact polypeptide strands, offering new possibilities for understanding complex biological processes and diseases.

Proteins are the molecular machines that drive nearly all functions in living organisms, from muscle movement to immune response. Proteins can exist in many different forms, known as proteoforms, which are created through slight variations such as mutations or post-translational modifications. These subtle changes can have a profound impact not only on protein function but also on disease development, including cancer, Alzheimer's, and autoimmune disorders. Although understanding these variations in full-length proteins is critical for deciphering their roles in health and disease, a protein’s complex structure has so far made it difficult to analyse at single molecule resolution.

In a new study, 'Multi-pass, single-molecule nanopore reading of long protein strands', a research team at the University of Washington, led by Jeff Nivala, showcased an innovative approach using a protein unfoldase, ClpX, in combination with Oxford Nanopore’s sensing platform, to enable the individual protein molecules to pass through the nanopore while generating a readable signal. The method allows scientists to 'unzip' and read the protein one amino acid at a time— analogous to nanopore DNA sequencing, but now applied to proteins. Crucially, this method also enables researchers to reread the same protein strand multiple times, boosting the accuracy of the results and making it easier to detect small but significant differences between proteoforms.

'This study highlights the remarkable versatility of the Oxford Nanopore sensing platform,' said Lakmal Jayasinghe, SVP of R&D Biologics at Oxford Nanopore Technologies. 'Beyond its established use in sequencing DNA and RNA, the platform can now be adapted for novel applications such as protein sequencing. With its distinctive features including portability, affordability, and real-time data analysis, researchers can delve into proteomics at an unprecedented level by performing sequencing of entire proteins using the nanopore platform. Exciting developments lie ahead for the field of proteomics with this groundbreaking advancement.'

This method represents ‘proof of concept’ that will require further R&D before broader clinical application, however the implications of accurately identifying and characterising full-length proteins are significant, creating potential opportunities to uncover new protein markers for disease and guide the development of targeted drugs. It also opens up new possibilities in precision medicine, where in the future treatments could be tailored based on an individual’s unique protein profile.

'This technology integrates many of the essential features required to develop a transformative approach to protein sequencing,' said Nivala, faculty and co-director of the Molecular Information Systems Lab in the UW’s Paul G. Allen School of Computer Science & Engineering. 'It could lead to groundbreaking applications in disease characterization, drug discovery, and the study of cellular processes, offering a level of detail previously unattainable.'

In addition to its potential clinical applications, the technology could also be applied to the development of protein-based barcoding systems, allowing researchers to create libraries of protein sequences for use in advanced diagnostics, therapeutic monitoring, and more.