Discriminating Residue Substitutions in a Single Protein Molecule Using a Sub-nanopore

Abstract

It is now possible to create, in a thin inorganic membrane, a single, sub-nanometer-diameter pore (i.e., a sub-nanopore) about the size of an amino acid residue. To explore the prospects for sequencing protein with it, measurements of the force and current were performed as two denatured histones, which differed by four amino acid residue substitutions, were impelled systematically through the sub-nanopore one at a time using an atomic force microscope. The force measurements revealed that once the denatured protein, stabilized by sodium dodecyl sulfate (SDS), translocated through the sub-nanopore, a disproportionately large force was required to pull it back. This was interpreted to mean that the SDS was cleaved from the protein during the translocation. The force measurements also exposed a dichotomy in the translocation kinetics: either the molecule slid nearly frictionlessly through the pore or it slipped-and-stuck. When it slid frictionlessly, regardless of whether the molecule was pulled N-terminus or C-terminus first through the pore, regular patterns were observed intermittently in the force and blockade current fluctuations that corresponded to the distance between stretched residues. Furthermore, the amplitude of the fluctuations in the current blockade were correlated with the occluded volume associated with the amino acid residues in the pore. Finally, a comparison of the patterns in the current fluctuations associated with the two practically identical histones supported the conclusion that a sub-nanopore was sensitive enough to discriminate amino acid substitutions in the sequence of a single protein molecule by measuring volumes of 0.1 nm per read.

Beyond mass spectrometry, the next step in proteomics

“…The problem with sensitivity hobbling MS can be resolved using a tool of a different stripe, i.e., a nanometer-diameter pore or nanopore through a thin membrane (74)(75)(76)(77)(78). A nanopore is the ultimate analytical tool with extreme single-molecule sensitivity derived from its minuscule volume.…”

“…However, the "jamming" of AAs in the pore checks the possibility of determining the AA sequence. On the other hand, using a subnanometer-diameter pore, i.e., a subnanopore, through a thin membrane with a yoctoliter sensing volume, the size of the single-AA residues comprising a single denatured protein can be directly read by measuring the blockade current when the molecule is stretched and forced through it (75)(76)(77).…”

“…Using this strategy, a sensitivity of V mol /V pore = 1.2 × 10 −9 has been achieved detecting 50-nm-diameter on July 4, 2020 http://advances.sciencemag.org/ Downloaded from nanoparticles in a micropore (118), but the sensitivity is generally compromised by noise and biofouling (clogging) as the pore diameter shrinks (119). Strategies have been developed to rescue the sensitivity that modulate the blockade current to find signal buried in the noise (118)(119)(120) and preclude biofouling by protein adhering to the pore (74,76,112).…”

“…Second, regardless of the 2D material, the peptide first collapses onto the membrane and is then impelled by an electric force unidirectionally and stepwise through the pore with a velocity that depends on the electric field and/or hydrostatic pressure gradients and water flow. The kinetics of a protein translocating through a pore actually can have two aspects to it: It can either slip-and-stick, unbinding and then binding again to the membrane, or it can slide nearly frictionless through the pore (76). According to MD, the stepwise motion yielded fluctuations in the blockade current that informed on the fragment of the peptide in the pore.…”

“…It is now technologically within our grasp to fix these problems by creating a subnanopore spanning a thin amorphous inorganic membrane. A subnanopore can be created by sputtering with a tightly focused, high-energy electron beam in a STEM through an amorphous silicon nitride membrane nominally 10 nm thick (75)(76)(77)(78)147). Pores made this way generally have a negatively charged surface and can be so small that only single dehydrated cations permeate across the membrane through them (Fig.…”