FraC nanopores with adjustable diameter identify the mass of opposite-charge peptides with 44 dalton resolution

Abstract

A high throughput single-molecule method for identifying peptides and sequencing proteins based on nanopores could reduce costs and increase speeds of sequencing, allow the fabrication of portable home-diagnostic devices, and permit the characterization of low abundance proteins and heterogeneity in post-translational modifications. Here we engineer the size of Fragaceatoxin C (FraC) biological nanopore to allow the analysis of a wide range of peptide lengths. Ionic blockades through engineered nanopores distinguish a variety of peptides, including two peptides differing only by the substitution of alanine with glutamate. We also find that at pH 3.8 the depth of the peptide current blockades scales with the mass of the peptides irrespectively of the chemical composition of the analyte. Hence, this work shows that FraC nanopores allow direct readout of the mass of single peptide in solution, which is a crucial step towards the developing of a real-time and single-molecule protein sequencing device.

Fig. 1

Preparation and characterization of type I, type II, and type III fragaceatoxin C (FraC) nanopores. a Cut through of a surface representation of wild-type FraC (Wt-FraC) oligomer (PDB: 4TSY) colored according to the vacuum electrostatic potential as calculated by PyMOL. One protomer is shown as a carton presentation with tryptophans 112 and 116 displayed as spheres. b Percentage of the distribution of type I, type II, and type III for Wt-FraC, W112S-FraC, W116S-FraC, and W112S-W116S-FraC at pH 7.5 and 4.5. c IV curves of type II nanopores formed by Wt-FraC, W116S-FraC, and W112S-W116S-FraC at pH 4.5. d Single nanopore conductance of W116S-FraC in 1 M KCl at pH 4.5 and –50 mV. e Typical current traces for the three nanopore types of W116S-FraC in 1 M KCl at pH 4.5 under –50 mV applied potential. f Reversal potentials measured under asymmetric condition of KCl (1960 mM cis, 467 mM trans) at pH 4.5 for the three W116S-FraC nanopore types. The ion selectivity was calculated using the Goldman–Hodgkin–Katz equation (Eq.  1). g Molecular models of the three type FraC nanopores constructed from the FraC crystals structure using the symmetrical docking function of Rosetta. The diameters were measured from the distance between opposite side chains of D10 and include the van der Waals radii of the atoms. The electrophysiology recordings were performed with a 10 kHz sampling and a 2 kHz Bessel filter. The error bars and color shadow in the I–V curves are standard deviations from at least three repeats

Fig. 2

Discrimination of angiotensin peptides using type II W116S-fragaceatoxin C (FraC) nanopores at pH 4.5. a Peptide sequences of angiotensin I (Ang I), angiotensin II (Ang II), angiotensin III (Ang III), and angiotensin IV (Ang IV) and typical blockades provoked by the four angiotensin peptides measured at −30 mV. b–e Color density plot of the I_ex% versus the standard deviation of the current amplitude for angiotensin I, II, III, and IV, respectively. f Discrimination of four angiotensin peptides in a mixture. Peptides were added into the cis chamber and measured at −30 mV. Standard deviations were calculated from at least three independent repeats. Color density plots were created using Origin

Fig. 3

Discrimination of peptides differing by a single amino acid using type II W116S-fragaceatoxin C (FraC) at pH 4.5. a Peptide sequences of angiotensin II, and A with typical blockades provoked by the two angiotensin peptides measured at −30 mV applied bias. b, c Color density plot of the I_ex% versus the standard deviation of the current amplitude for angiotensin II, and A, respectively. d Separation of angiotensin II and A in a mixture. Peptides were added into the cis chamber and measured under −30 mV. Standard deviations were calculated from at least three independent repeats

Fig. 4

Recognition of peptides with different chemical composition at pH 4.5. On the top graph is the relation between the molecular weight (M.W.) or volume of the peptide and the I_ex%. The bottom figure shows the sensing volume of type I wild-type fragaceatoxin C (Wt-FraC) (a), type II W116S-FraC (b), and type III W112S-W116S-FraC (c) nanopores. The solid line represents a second order polynomial fitting in a, b and a linear fitting in c, with the extrapolated value at 100% I_ex% corresponding to the volume of a peptide that would completely occupy the sensing volume of the nanopore. The latter is most likely constricted to the volume included between the constriction of the pore (aspartic acid 10) and the residues that lie one turn of a helix above the constriction (aspartic acid 17). The distances are measured from two opposing residues and include the van der Waals radii of the atoms. Current blockades were measured at −30 mV for type I and II pore, and at −50 mV for type III pore in 1 M KCl solutions. The error bars represent standard deviation from at least three repeats. Red circles highlight the two peptides that bare a negative charge at pH 4.5 (Table 1)

Fig. 5

A nanopore peptide mass identifier. a Top, sequence of the four peptides tested. The amino acids that have a positive charge are in blue and the acidic residues in red. Below, pH dependence of the I_ex% for the four peptides (cis) using type II W116S-fragaceatoxin C (FraC) nanopores under –30 mV applied potential. b Relationship between the I_ex% and the mass of peptides at pH 3.8. c Voltage dependence of c-Myc dwell times at different pHs. All electrophysiology measurements were carried out in 1 M KCl, 0.1 M citric acid. The charges of the peptides were calculated according to the pK_a for individual amino acids. Standard deviations were calculated from at least three independent repeats