Single-File Translocation Dynamics of SDS-Denatured, Whole Proteins through Sub-5 nm Solid-State Nanopores

Abstract

The ability to routinely identify and quantify the complete proteome from single cells will greatly advance medicine and basic biology research. To meet this challenge of single-cell proteomics, single-molecule technologies are being developed and improved. Most approaches, to date, rely on the analysis of polypeptides, resulting from digested proteins, either in solution or immobilized on a surface. Nanopore biosensing is an emerging single-molecule technique that circumvents surface immobilization and is optimally suited for the analysis of long biopolymers, as has already been shown for DNA sequencing. However, proteins, unlike DNA molecules, are not uniformly charged and harbor complex tertiary structures. Consequently, the ability of nanopores to analyze unfolded full-length proteins has remained elusive. Here, we evaluate the use of heat denaturation and the anionic surfactant sodium dodecyl sulfate (SDS) to facilitate electrokinetic nanopore sensing of unfolded proteins. Specifically, we characterize the voltage dependence translocation dynamics of a wide molecular weight range of proteins (from 14 to 130 kDa) through sub-5 nm solid-state nanopores, using a SDS concentration below the critical micelle concentration. Our results suggest that proteins' translocation dynamics are significantly slower than expected, presumably due to the smaller nanopore diameters used in our study and the role of the electroosmotic force opposing the translocation direction. This allows us to distinguish among the proteins of different molecular weights based on their dwell time and electrical charge deficit. Given the simplicity of the protein denaturation assay and circumvention of the tailor-made necessities for sensing protein of different folded sizes, shapes, and charges, this approach can facilitate the development of a whole proteome identification technique.

Keywords: SDS−protein complex; electrical charge deficit; electroosmotic force; protein translocation; single-molecule sensing; solid-state nanopores; voltage-driven translocation.

Figure 1

Characterization of SDS micelles and SDS-denatured BSA proteins translocations using sub-5 nm ssNPs. (a) Ion current traces at different protein and SDS concentrations show two distinct events amplitudes corresponding to SDS micelles and BSA proteins (BSA concentration 1–1.5 nM). (b) Event diagrams, shown as heatmaps, of the event amplitude (ΔI) vs dwell-time (log t_D) for different SDS and protein concentrations: (I) SDS only (700 μM), (II) SDS + BSA (350 μM/1 nM), (III) SDS + BSA (175 μM/1.5 nM). (c) Corresponding histograms of event amplitudes show two distinct peaks for SDS micelles (0.5–0.7 nA) and BSA proteins (1.1–1.3 nA).

Figure 2

Translocation dynamics of SDS-denatured carbonic anhydrase (CA) proteins in three different nanopore sizes. (a) Translocation events diagrams shown as heat-maps for CA proteins using three nanopore sizes (left to right): ∼3 nm, G = 7 ns; ∼4 nm, G = 12 nS; and ∼7 nm, G = 21 nS. In each case, the event amplitudes (ΔI) are shown vs the event dwell-times (log t_D). Typical events are displayed in insets, showing a clear shift toward longer events in the smaller nanopores, but similar event amplitudes. (b) Histograms of the electrical charge deficit (ECD) for the three experiments (shown in semilog scale). In all cases, the ECD histogram displays a prominent peak around 10⁶e and secondary peaks at 10^8.3e, 10⁷e, and 10^6.5e for the smaller and larger pores, respectively. Minor peaks at much lower ECD, possibly due to collisions, are also visible. Red curves are double Gaussian fits (see text).

Figure 3

Voltage dependence of translocation dynamics of SDS-denatured alpha-lactalbumin proteins. (A) Electrical charge deficit (ECD) vs voltage. Black circles represent the main peak, and red squares correspond to the secondary peak. The main ECD peak is nearly independent of voltage, whereas the secondary peak shows a mild decrease with voltage. (B) Dwell-time (t_d) vs voltage. Black circles correspond to events in the main peak, and red squares (inset) correspond to the secondary peak. The solid lines are decaying exponential fits. The experiments were performed in 10% glycerol (v/v), 0.4 M NaCl, 1× PBS, and 175 μM SDS.

Figure 4

SDS-PAGE analysis of six proteins used in this study compared with nanopore measurements. (a) PAGE analysis, from right to left: alpha-lactalbumin, carbonic anhydrase, ovalbumin, BSA, phosphorylase B, and spike protein were denatured and separated on a 4–20% Tris-glycine SDS-PAGE. The gel was fixed, stained with Flamingo fluorescent dye overnight, and imaged by the Pharos scanner (Bio-Rad). L denotes lanes with protein ladders (see Methods). (b) Dependence of the SDS-PAGE migration distance of the six proteins on their molecular weights. The solid line is an exponential fit. (c) Dependence of SDS-denatured protein translocation dwell-time on the PAGE migration distance. The solid line is a linear fit.

Figure 5

Translocation dynamics of SDS-denatured proteins as a function of molecular weight (M_w). (a) Characteristic dwell-times of the six proteins in the nanopore. The black solid circles represent the main peak (a, b), while the secondary peak is represented by red squares (a, b). We observe a linear increase of t_d on M_w for the main peak (black straight line fit) and a nonlinear dependence for the second group (red line is a guide for the eye). (b) Summary plot of the ECD as a function of M_w. The main ECD peak event (solid circle) shows a linear dependency on M_w, while the secondary peak event (red squares) exhibits a quadratic dependency. Solid lines are fits.

Figure 6

Model for elucidation of voltage-induced translocation of SDS/protein complexes through small solid-state nanopores. (a) Electrical charge deficit (ECD) for phosphorylase B, where two peaks can be distinguished clearly. The representative events corresponding to the first peak (represented in black color) show no sublevels in the ionic current trace during translocations. However, the event with the sublevel ionic current traces (red-colored events) corresponds to subtle secondary or tertiary motifs that exist during the protein translocations. (c) Schematic illustration for the two different scenarios. (b) Effect of SDS coating on the pore walls enhancing the EOF, resulting in the longer translocation time in comparison to a similar size dsDNA molecule.