Nanopore Sequencing Detects Protein Structures

By Allison Proffitt 

August 16, 2023 | University of Oxford researchers have detected protein structure modifications using nanopore technology. The method, published in Nature Nanotechnology, (DOI: https://doi.org/10.1038/s41565-023-01462-8) identified phosphorylation, glutathionylation, and glycosylation at the single-molecule level for protein chains over 1,200 residues long. 

Human cells contain approximately 20,000 protein-encoding genes. However, the actual number of proteins observed in cells is far greater with over 1 million different structures known. These variants are generated through post-translational modification (PTM), introducing structural changes such as the addition of chemical groups or carbohydrate chains to the individual amino acids that make up proteins, resulting in hundreds of possible variations for the same protein chain.   

These variants play pivotal roles in biology by enabling precise regulation of complex biological processes within individual cells. Mapping this variation would uncover a wealth of valuable information that could revolutionize our understanding of cellular functions. But to date, the ability to produce comprehensive protein inventories has remained an elusive goal.  

To overcome this, the team used nanopore DNA/RNA sequencing technology. In their approach, a directional flow of water captures and unfolds proteins into linear chains that are fed through pores just wide enough for a single amino acid molecule to pass. Structural variations are identified by measuring changes in an electrical current applied across the nanopore. Different molecules cause different disruptions in the current, giving them a unique signature. 

There are some unique challenges to using nanopores for proteins, the authors highlighted. First, proteins must be linear, and second, “unlike DNA or RNA, polypeptides have a low-density and heterogeneous distribution of charge along their chains, which renders electrophoresis inapplicable as a means of translocation,” the authors—one of whom is Professor Hagan Bayley (Department of Chemistry, University of Oxford), co-founder of Oxford Nanopore Technologies—write in the paper. They needed to find another way to drive the polypeptide chain through the pore.  

They chose a method that does not require the use of labels, enzymes, or additional reagents. “We use strong electro-osmosis directly attributable to the charged side chains in an engineered [staphylococcal α-hemolysin] (αHL) pore to capture long underivatized polypeptides and detect modifications within them as they are unfolded and translocated,” the authors write.  

Next Steps 

“Our strategy will be readily transferable to nanopore sequencing devices (for example, the MinION) for highly parallel PTM profiling, which will be useful for producing inventories of full-length human proteoforms, which are ~500 [amino acids] in median length,” the authors observe. This could facilitate point-of-care diagnostics, enabling the personalized detection of specific protein variants associated with diseases including cancer and neurodegenerative disorders. 

There are persistent challenges, though, and the authors are clear about that. Proteins differ in ease of unfolding, so there may not be universal conditions for translocation. Capture rate will also probably differ between proteins. Some PTMs are particularly compact (for example, methylation), and in those cases, the process may require ligand-assisted detection with antibodies or chemical binders.  

But the approach still shows great promise, the study authors believe, enabling huge, new datasets that could reveal unknown aspects of the biology of cells and tissues.  

Bayley commented in a press release about the work: “The ability to pinpoint and identify post-translational modifications and other protein variations at the single-molecule level holds immense promise for advancing our understanding of cellular functions and molecular interactions. It may also open new avenues for personalized medicine, diagnostics, and therapeutic interventions.”