Calling the Amino Acid Sequence of a Protein/peptide from the Nanospectrum Produced by a Sub-nanometer Diameter Pore

The blockade current that develops when a protein translocates across a thin membrane through a sub-nanometer diameter pore informs with extreme sensitivity on the sequence of amino acids that constitute the protein. The current blockade signals measured during the translocation are called a nanospectrum of the protein. Whereas mass spectrometry (MS) is still the dominant technology for protein identification, it suffers limitations. In proteome-wide studies, MS identifies proteins by database search but often fails to provide high protein sequence coverage. It is also not very sensitive requiring about a femtomole for protein identification. Compared with MS, a sub-nanometer diameter pore (i.e. a sub-nanopore) directly reads the amino acids constituting a single protein molecule, but efficient computational tools are still required for processing and interpreting nanospectra. Here, we delineate computational methods for processing sub-nanopore nanospectra and predicting theoretical nanospectra from protein sequences, which are essential for protein identification.