Reading the primary structure of a protein with 0.07 nm3 resolution using a subnanometre-diameter pore

Abstract

The primary structure of a protein consists of a sequence of amino acids and is a key factor in determining how a protein folds and functions. However, conventional methods for sequencing proteins, such as mass spectrometry and Edman degradation, suffer from short reads and lack sensitivity, so alternative approaches are sought. Here, we show that a subnanometre-diameter pore, sputtered through a thin silicon nitride membrane, can be used to detect the primary structure of a denatured protein molecule. When a denatured protein immersed in electrolyte is driven through the pore by an electric field, measurements of a blockade in the current reveal nearly regular fluctuations, the number of which coincides with the number of residues in the protein. Furthermore, the amplitudes of the fluctuations are highly correlated with the volumes that are occluded by quadromers (four residues) in the primary structure. Each fluctuation, therefore, represents a read of a quadromer. Scrutiny of the fluctuations reveals that the subnanometre pore is sensitive enough to read the occluded volume that is related to post-translational modifications of a single residue, measuring volume differences of ∼0.07 nm, but it is not sensitive enough to discriminate between the volumes of all twenty amino acids.

Single-molecule protein identification by sub-nanopore sensors

“…The details regarding the experiments and methods used to acquire electrical current blockade signals from the translocation of single protein molecules through sub-nanopores have been described elsewhere [12]. To summarize, first, a pore with sub-nanometer cross-section was sputtered through thin silicon nitride membrane supported on a silicon chip using a tightly focused, high-energy electron beam in a scanning transmission electron microscope (Fig 1).…”

“…It was determined that a lower transmembrane bias voltage improved the signal-to-noise ratio (SNR) and lengthened the median duration of the blockades, but it also increased the propensity for the pore to clog. Multi-level events associated with residual native protein structure or multiple molecules competing for the pore were occasionally observed, but were manually culled from the data pre- analysis [12]. …”

“…Experimental analysis of peptides with post-translational modifications and mutations [12] revealed changes in the specific regions of the recorded signal traces, that corresponded to approximately four amino acids in length. In addition, simulations of the electric field in a 0.5x0.5 nm 2 diameter, 8 nm thick pore in an SiN membrane indicated that the vast majority of the field was confined within 1.5 nm of the pore near the waist at the center of the membrane, which gives roughly the same estimate of the number of amino acids.…”

“…Recently, the sequence of amino acids in a denatured protein were read with limited resolution using a sub-nanometer-diameter pore, sputtered through a thin silicon nitride membrane [12]. Protein translocations through the pore modulated the measured ionic current, which was correlated with the volumes of amino adids in the proteins.…”

“…However, it is about 100-fold less sensitive than bottom-up mass spectrometry, which can be used to detect attomoles of material [13]. In stark contrast, a sub-nanopore has been used to discriminate residue substitutions in a single molecule with low fidelity [12]. …”