Single-Molecule Protein Sequencing Using Biological Nanopores

The next wave of research in bioinformatics and fundamental biology will revolve around sequencing the enormous and dynamic variations in the proteome. A method for sequencing proteins and detecting PTMs at the single-molecule level would be revolutionary for proteomics research. We report proof-of-concept and initial results for a new single-molecule protein sequencing technique. We use a typical enzyme-controlled biological nanopore sequencing setup, with the addition of a peptide fragment covalently linked to the end of the DNA strand. The nanopore sequencer first reads the DNA, then the peptide fragment as the DNA-peptide conjugate is pulled up through the nanopore. The readout is then used to decode the peptide sequence, map substitutions and post-translation modifications, or identify the peptide from a library of proteins. Similarly to nanopore DNA sequencing, this technique is in principle capable of single-molecule measurements, high throughput, low experimental overhead and minimal sample preparation, and sensitivity to changes in individual monomers.