Abstract
Direct protein sequencing technologies with improved sensitivity and throughput are still needed. Here, we propose an alternative method for peptide sequencing based on enzymatic cleavage and host–guest interaction-assisted nanopore sensing. We serendipitously discovered that the identity of any proteinogenic amino acid in a particular position of a phenylalanine-containing peptide could be determined via current blockage during translocation of the peptide through α-hemolysin nanopores in the presence of cucurbit[7]uril. Building upon this, we further present a proof-of-concept demonstration of peptide sequencing by sequentially cleaving off amino acids from C terminus of a peptide with carboxypeptidases, and then determining their identities and sequence with a peptide probe in nanopore. With future optimization, our results point to a different way of nanopore-based protein sequencing.
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Data availability
Data supporting the findings of this study are given in the main text and the Supplementary Information. Source data have been deposited in the Zenodo database under accession code: https://doi.org/10.5281/zenodo.8358079. Source data are provided with this paper.
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Acknowledgements
This project was funded by the National Natural Science Foundation of China (nos. 22025407 and 21974144 to H.-C.W.; 21874136 to L.L.) and Institute of Chemistry, Chinese Academy of Sciences. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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Authors and Affiliations
Contributions
Y.Z. and Y.Y. performed amino acid discrimination experiments. Y.Z., Y.Y. and Z.L. performed peptide sequencing experiments. Y.Z., Y.Y. and Z.K. performed protein mutation experiments. Z.Y., Y.Y., L.L. and H.-C.W. performed data analysis. Z.Y., L.L. and H.-C.W. conceived the project, designed the experiments and wrote the paper.
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Competing interests
H.-C.W., Y.Z., Y.Y. and Z.L. have filed patents describing the strategy for the nanopore-based peptide sequencing. All other authors have no competing interests.
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Nature Methods thanks Abdelghani Oukhaled and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Arunima Singh, in collaboration with the Nature Methods team.
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Extended data
Extended Data Fig. 1 Successive addition and identification of FGXD8 in WT aHL nanopore.
(a) Current traces showing characteristic current events generated by the translocation of 12 different FGXD8⊂CB[7] peptides through WT αHL. (b–m) Histogram of I/I0 histogram distribution of recorded events after stepwise addition of different FGXD8. All data were acquired in the buffer of 3.6 M KCl, 10 mM citric acid, pH 5.0, with the transmembrane potential held at +200 mV. The final concentration of each peptide in cis compartment is 150 nM. Note that the count of FGGD8 was multiplied by a factor of 0.3 for making graphs due to its high capture rate.
Extended Data Fig. 2 Using (M113F)7 aHL to distinguish FGXD8 peptides that exhibited overlapping scatter-plots with WT aHL.
The scatter-plot of current blockades versus event durations recorded from an equimolar mixture of (a,b) FGID8 and FGLD8, (c,d) FGFD8 and FGCD8, (e,f) FGAD8 and FGWD8, (g,h) FGSD8 and FGPD8, (i,j) FGDD8 and FGQD8, (k,l) FGQD8 and FGHD8 with WT and M113F αHL, respectively. Well-separated distributions were obtained for each pair using M113F αHL. All data were acquired in 3.6 M KCl, 10 mM citric acid buffer, at pH 5.0, with the transmembrane potential held at +200 mV. The final concentration of FGXD8⊂CB[7] is 400 nM.
Extended Data Fig. 3 Typical scatter-plot of dwell time versus I/I0 and histograms of the current blockades I/I0 generated by the translocation of FGGC(P)D8⊂CB[7], FGGC(K)D8⊂CB[7], FGGC(H)D8⊂CB[7] and FGGC(W)D8⊂CB[7] through WT αHL.
(a, b) FGGC(P)D8⊂CB[7]. (c, d) FGGC(K)D8⊂CB[7]. (e, f) FGGC(H)D8⊂CB[7]. (g, h) FGGC(W)D8⊂CB[7]. We noted that all the four coupled peptides produced two peaks when translocated through αHL. For FGGC(P)D8, the result demonstrates that the relative residual current is sensitive to the spatial orientation of the peptide due to the presence of the pyrrolidine ring. For FGGC(K)D8, the lysine has two different reaction sites (α- or ε-amino groups) when it is linked to the probe peptide, and therefore it is possible that the two current blockades are caused by two different FGGC(K)D8 translocated through αHL nanopore. For FGGC(H)D8⊂CB[7] and FGGC(W)D8⊂CB[7], it is very likely that the side chains of H and W can also interact with CB[7] to produce an extra current blockade. All data were acquired in 3.6 M KCl, 10 mM citric acid buffer, at pH 5.0, with the transmembrane potential held at +200 mV. The final concentration of FGGC(G)D8 is 400 nM.
Extended Data Fig. 4 Scatter-plots of discrimination of amino acids D, N, Q, E, I, L with WT and mutant αHL nanopore experiments.
(a) Q and I with (M113G)7 αHL nanopore. (b) I and L with (M113G)7 αHL nanopore. (c) L and E at pH 4.0 with (M113G)7 αHL nanopore. (d) E and N at pH 4.0 with (M113G)7 αHL nanopore. (e) N and D at pH 4.0 with (M113G)7 αHL nanopore. (f) D and Q at pH 4.0 with (M113G)7 αHL nanopore. (g) Q and L with (M113R)7 αHL nanopore. All data were acquired in the buffer of 3.6 M KCl, 10 mM citric acid, pH 5.0, with the transmembrane potential held at +200 mV.
Extended Data Fig. 5 Discrimination of amino acids A, S, T, V with WT and mutant αHL nanopore experiments.
(a) Discrimination process for amino acid A, S, T, V using WT and mutant αHL. The lines are the Gaussian fit to the histograms of I/I0 values of the amino acids A, S, T, V obtained from WT and mutant αHL nanopore measurements. (b-e) Scatter-plots of discrimination of amino acids A, S, T, V with WT and mutant αHL nanopore measurements. All data were acquired in the buffer of 3.6 M KCl, 10 mM citric acid, pH 5.0, with the transmembrane potential held at +200 mV.
Extended Data Fig. 6 Discrimination of amino acids F and M with WT and mutant αHL nanopore experiments.
(a) Discrimination process for amino acid M and F using WT and mutant αHL. The lines are the Gaussian fit to the histograms of I/I0 values of the amino acids F, M obtained from WT and (M113G)7 αHL nanopore measurements. (b) The scatter-plots of recognition process of indistinguishable amino acid FM using WT and (M113G)7 αHL. All data were acquired in the buffer of 3.6 M KCl, 10 mM citric acid, pH 5.0, with the transmembrane potential held at +200 mV.
Extended Data Fig. 7 The influence of digestion time of CPA and CPB on protein sequencing.
Carboxypeptidase digestion was performed at 37 °C for (a-c) 20 s, (d-f) 10 min, (g-i) 30 min, (j-l) 60 min, (m-o) 120 min. All data were acquired in the buffer of 3.6 M KCl, 10 mM citric acid, pH 5.0, with the transmembrane potential held at +200 mV. Data are presented as mean values ± SD. Number of individual experiments n = 3.
Extended Data Fig. 8 Sequencing pep2 with FGGCD8 using a nanopore.
(a) Scatter-plot of dwell time versus I/I0 generated by the translocation of FGGC(X)D8 through WT αHL. (b) Integral area of I/I0 generated by FGGC(X)D8. (c) Normalized ratios of the integral area of I/I0 for each FGGC(X)D8 which are used to determine the sequence of the amino acids in the original model peptides. All data were acquired in the buffer of 3.6 M KCl, 10 mM citric acid, pH 5.0, with the transmembrane potential held at +200 mV. Data are presented as mean values ± SD. Number of individual experiments n = 3.
Extended Data Fig. 9 The detailed data acquisition and analysis of the stepwise enzymatic digestion of pep3 in Fig. 4e.
The peptide was digested by either dilute CPA or CPB during each step. (a-c) Step 1: pep3 was digested with dilute CPA (0.12 U/mL) for 8 min. Then the solution was ultra-centrifuged to isolate the cleaved amino acids and the remaining peptides. The amino acids were taken to coupling reactions and subsequent nanopore identification. Amino acids F and Q were identified in this step. (d-f) Step 2: The remaining peptide from Step 1 was digested with dilute CPA for another 8 min. The procedures of ultra-centrifugation, coupling reaction and nanopore identification were carried out exactly the same as in Step 1. Amino acids V and A were identified in this step. Then the digestion was repeated again. It was found that no new amino acid signals were detected. In this case, the peptide was digested with CPA (1.0 U/mL) for 2 h to completely cleave off F, Q, V and A. (g-i) Step 3: The remaining peptide from Step 2 was digested with CPB (2 U/mL) for 14 min. Amino acid R was identified in this step. (j-l) Step 4: The remaining peptide from Step 3 with dilute CPA for 8 min. Amino acids Y and I were identified in this step. (m-o) Step 5: The remaining peptide from Step 4 with dilute CPA for 8 min. Amino acids M and G were identified in this step. Another complete digestion was conducted in this step. (p-r) Step 6: The remaining peptide from Step 5 with CPB for 14 min. Another R was identified in this step. All data were acquired in the buffer of 3.6 M KCl, 10 mM citric acid, pH 5.0, with the transmembrane potential held at +200 mV. Number of individual experiments n = 3. Data are presented as mean values ± SD.
Extended Data Fig. 10 Discrimination of cleaved amino acids F and M, I and Q from the peptide with (M113G)7 αHL nanopore in Fig. 4e.
(a-b) The scatter-plots and histograms of I/I0 values of events in Step 1 enzymatic digestion. (c-d) The scatter-plots and histograms of I/I0 values of events in Step 4 enzymatic digestion. (e-f) The scatter-plots and histograms of I/I0 values of events in Step 5 enzymatic digestion. All data were acquired in the buffer of 3.6 M KCl, 10 mM citric acid, pH 5.0, with the transmembrane potential held at +200 mV.
Supplementary information
Supplementary Information
Supplementary Figures 1–14 and Supplementary Tables 1 and 2.
Supplementary Data 1
Statistical analysis of the events generated by the translocation of FGGD8⊂CB[7] through WT αHL.
Supplementary Data 2
Investigation of the influence of the applied potentials on type I events during the translocation of FGGD8⊂CB[7] through WT αHL nanopore.
Supplementary Data 3
Condition screening for amino acid X discrimination.
Supplementary Data 4
Typical histogram of the current blockades I/I0 generated by the translocation of FGXD8⊂CB[7] through WT αHL nanopore.
Supplementary Data 5
Typical histogram of the current blockades I/I0 generated by the translocation of FGXD8⊂CB[7] through (M113F)7 αHL.
Supplementary Data 6
Mass spectroscopic characterization of probe peptides FGCD8, FGGCD8, FGGGCD8 coupled with G and R.
Supplementary Data 7
Mass spectroscopic characterization of 18 different coupled FGGC(X)D8 peptides.
Supplementary Data 8
Coupling of amino acid C to FGGCD8⊂CB[7] and its translocation through WT αHL.
Supplementary Data 9
Mass spectra resulting from CPA and CPB digestion of the two model peptides.
Supplementary Data 10
Event frequency of the translocation of FGGC(G)D8⊂CB[7] through WT αHL versus the concentrations of FGGC(G)D8.
Supplementary Data 11
Determination of the recognition site on the peptide probe for amino acid discrimination.
Supplementary Data 12
Screening of the coupling peptide probe for amino acid discrimination.
Source data
Source Data Fig. 1
Identification of the 20 proteinogenic amino acids using the FGXD8 probe.
Source Data Fig. 2
Correlation of the mean I/I0 values of the translocation of FGXD8⊂CB[7] through WT αHL with the properties of amino acid X.
Source Data Fig. 3
Identification of the 20amino acids using the FGGCD8 probe.
Source Data Fig. 4
The proof-of-concept demonstration of peptide sequencing with FGGCD8 using a nanopore.
Source Data Extended Data Fig. 1
Successive addition and identification of FGXD8 in WT αHL nanopore.
Source Data Extended Data Fig. 2
Using (M113F)7 αHL to distinguish FGXD8 peptides that exhibited overlapping scatter-plots with WT αHL.
Source Data Extended Data Fig. 3
Typical scatter-plot of dwell time versus I/I0 and histograms of the current blockades I/I0 generated by the translocation of FGGC(P)D8⊂CB[7], FGGC(K)D8⊂CB[7], FGGC(H)D8⊂CB[7] and FGGC(W)D8⊂CB[7] through WT αHL.
Source Data Extended Data Fig. 4
Scatter-plots of discrimination of amino acids D, N, Q, E, I, L with WT and mutant αHL nanopore experiments.
Source Data Extended Data Fig. 5
Discrimination of amino acids A, S, T, V with WT and mutant αHL nanopore experiments.
Source Data Extended Data Fig. 6
Discrimination of amino acids F and M with WT and mutant αHL nanopore experiments.
Source Data Extended Data Fig. 7
The influence of digestion time of CPA and CPB on protein sequencing.
Source Data Extended Data Fig. 8
Sequencing pep2 with FGGCD8 using a nanopore.
Source Data Extended Data Fig. 9
The detailed data acquisition and analysis of the stepwise enzymatic digestion of pep3 in Fig. 4e.
Source Data Extended Data Fig. 10
Discrimination of cleaved amino acids F and M, I and Q from the peptide with (M113G)7 αHL nanopore in Fig. 4e.
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Zhang, Y., Yi, Y., Li, Z. et al. Peptide sequencing based on host–guest interaction-assisted nanopore sensing. Nat Methods (2023). https://doi.org/10.1038/s41592-023-02095-4
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DOI: https://doi.org/10.1038/s41592-023-02095-4
