Calling the amino acid sequence of a protein/peptide from the nanospectrum produced by a sub-nanometer diameter pore

Abstract

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.

Horizontal detection of post-translational modifications to an amino acid with a nanopore based on analyte volume and translocation-time

“…The intrusion of the analyte results in a decrease or blockade in the normal ionic current. The size of this blockade is roughly proportional to the volume of the analyte [22,23]. Additionally the time taken by an analyte to translocate through the pore can be used to distinguish among different analytes.…”

Decoding Proteoforms with Single Acid Resolution Using a Sub-nanometer Diameter Pore

“…a sub-nanopore , immersed in electrolyte and measuring the pore current. 13–17 As the denatured proteoform, agglomerated with s odium d odecyl s ulfate (SDS), moves processively through the sub -nanopore, the SDS is stripped from the proteoform and the ion flow around it fluctuates reproducibly in relation to the acids in the pore waist. If the pore current is sampled at a high enough rate (Ms/sec) in line with the acid velocity, and the electric field in the pore is confined to a sub -nanometer extent near the waist, then the fluctuations in the pore current inform on a single acid and the pattern of fluctuations reflects the AA sequence.…”

“…18,19 The fluctuation patterns are identified using an a ffinity p ropagation (AP) algorithm without reference to any model for the peptide structure, unlike earlier work that compared the fluctuation patterns to a volume model of the sequence. 17 This distinction is important because it open a window into the identification of a protein in a mixture. Third, to call acids from the fluctuation patterns in the pore current, m olecular d ynamic (MD) simulations, which is accomplished with atomic precision, are indispensable.…”

“…Thus, MD can be used as basis for interpreting the fluctuation pattern with confidence, which is unprecedented. Finally, after accounting for differences in the acid mobility by using d ynamical t ime w arping (DTW) to align time sequence, 17,20–22 the empirical fluctuations in the pore current are nearly perfectly correlated to the MD simulations of the ion flow around a single acid translocating through the pore. The correlation between the aligned time sequence and the MD simulation of the current means that the acid sequence can be “read” with single acid resolution from the fluctuations with only a minimal amount of material—a few hundred molecules—required to identify a peptide.…”

“…A sub -nanopore was sputtered through the a-Si membrane using a tightly focused, high energy (300 kV) electron beam formed in a s canning t ransmission e lectron m icroscope (STEM). 17 Immediately after sputtering, the pore was visualized in situ with TEM to reveal a 1.5 nm-diameter at the waist defined by the shot noise (Fig. 1c).…”