Abstract
Single molecule protein sequencing would tremendously impact in proteomics and human biology and it would promote the development of novel diagnostic and therapeutic approaches. However, its technological realization can only be envisioned, and huge challenges need to be overcome. Major difficulties are inherent to the structure of proteins, which are composed by several different amino-acids. Despite long standing efforts, only few complex techniques, such as Edman degradation, liquid chromatography and mass spectroscopy, make protein sequencing possible. Unfortunately, these techniques present significant limitations in terms of amount of sample required and dynamic range of measurement. It is known that proteins can distinguish closely similar molecules. Moreover, several proteins can work as biological nanopores in order to perform single molecule detection and sequencing. Unfortunately, while DNA sequencing by means of nanopores is demonstrated, very few examples of nanopores able to perform reliable protein-sequencing have been reported so far. Here, we investigate, by means of molecular dynamics simulations, how a re-engineered protein, acting as biological nanopore, can be used to recognize the sequence of a translocating peptide by sensing the “shape” of individual amino-acids. In our simulations we demonstrate that it is possible to discriminate with high fidelity, 9 different amino-acids in a short peptide translocating through the engineered construct. The method, here shown for fluorescence-based sequencing, does not require any labelling of the peptidic analyte. These results can pave the way for a new and highly sensitive method of sequencing.
References
Steen, H.; Mann, M. The abc’s (and xyz’s) of peptide sequencing. Nat. Rev. Mol. Cell Biol. 2004, 5, 699–711.
Shimonishi, Y.; Hong, Y. M.; Kitagishi, T.; Matsuo, T.; Matsuda, H.; Katakuse, I. Sequencing of peptide mixtures by edman degradation and field-desorption mass spectrometry. Eur. J. Biochem. 1980, 112, 251–264.
Domon, B.; Aebersold, R. Options and considerations when selecting a quantitative proteomics strategy. Nat. Biotechnol. 2010, 28, 710–721.
Restrepo-Pérez, L.; Joo, C.; Dekker, C. Paving the way to single-molecule protein sequencing. Nat. Nanotechnol. 2018, 13, 786–796.
Ameur, A.; Kloosterman, W. P.; Hestand, M. S. Single-molecule sequencing: Towards clinical applications. Trends Biotechnol. 2019, 37, 72–85.
Nivala, J.; Marks, D. B.; Akeson, M. Unfoldase-mediated protein translocation through an α-hemolysin nanopore. Nat. Biotechnol. 2013, 31, 247–250.
Kennedy, E.; Dong, Z. X.; Tennant, C.; Timp, G. Reading the primary structure of a protein with 0.07 nm3 resolution using a subnanometre-diameter pore. Nat. Nanotechnol. 2016, 11, 968–976.
Kolmogorov, M.; Kennedy, E.; Dong, Z. X.; Timp, G.; Pevzner, P. A. Single-molecule protein identification by sub-nanopore sensors. PLoS Comput. Biol. 2017, 13, e1005356.
Wilson, J.; Sloman, L.; He, Z. R.; Aksimentiev, A. Graphene nanopores for protein sequencing. Adv. Funct. Mater. 2016, 26, 4830–4838.
Asandei, A.; Rossini, A. E.; Chinappi, M.; Park, Y.; Luchian, T. Protein nanopore-based discrimination between selected neutral amino acids from polypeptides. Langmuir 2017, 33, 14451–14459.
Farimani, A. B.; Heiranian, M.; Aluru, N. R. Identification of amino acids with sensitive nanoporous MoS2: Towards machine learning-based prediction. npj 2D Mater. Appl. 2018, 2, 14.
Ohayon, S.; Girsault, A.; Nasser, M.; Shen-Orr, S.; Meller, A. Simulation of single-protein nanopore sensing shows feasibility for whole-proteome identification. PLoS Comput. Biol. 2019, 15, e1007067.
Rosen, C. B.; Rodriguez-Larrea, D.; Bayley, H. Single-molecule site-specific detection of protein phosphorylation with a nanopore. Nat. Biotechnol. 2014, 32, 179–181.
Van Ginkel, J.; Filius, M.; Szczepaniak, M.; Tulinski, P.; Meyer, A. S.; Joo, C. Single-molecule peptide fingerprinting. Proc. Natl. Acad. Sci. USA 2018, 115, 3338–3343.
Zhao, Y. N.; Ashcroft, B.; Zhang, P. M.; Liu, H.; Sen, S. M.; Song, W. S.; Im, J.; Gyarfas, B.; Manna, S.; Biswas, S. et al. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling. Nat. Nanotechnol. 2014, 9, 466–473.
Ohshiro, T.; Tsutsui, M.; Yokota, K.; Furuhashi, M.; Taniguchi, M.; Kawai, T. Detection of post-translational modifications in single peptides using electron tunnelling currents. Nat. Nanotechnol. 2014, 9, 835–840.
Ouldali, H.; Sarthak, K.; Ensslen, T.; Piguet, F.; Manivet, P.; Pelta, J.; Behrends, J. C.; Aksimentiev, A.; Oukhaled, A. Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore. Nat. Biotechnol. 2020, 38, 176–181.
Swaminathan, J.; Boulgakov, A. A.; Marcotte, E. M. A theoretical justification for single molecule peptide sequencing. PLoS Comput. Biol. 2015, 11, e1004080.
Venkatesan, B. M.; Bashir, R. Nanopore sensors for nucleic acid analysis. Nat. Nanotechnol. 2011, 6, 615–624.
Hu, R.; Tong, X.; Zhao, Q. Four aspects about solid-state nanopores for protein sensing: Fabrication, sensitivity, selectivity, and durability. Adv. Healthc Mater., in press, DOI: https://doi.org/10.1002/adhm.202000933.
Huang, J. A.; Mousavi, M. Z.; Giovannini, G.; Zhao, Y. Q.; Hubarevich, A.; Soler, M. A.; Rocchia, W.; Garoli, D.; De Angelis, F. Multiplexed discrimination of single amino acid residues in polypeptides in a single SERS hot spot. Angew. Chem., Int. Ed. 2020, 59, 11423–11431.
Sheinerman, F. B.; Norel, R.; Honig, B. Electrostatic aspects of protein-protein interactions. Curr. Opin. Struct. Biol. 2000, 10, 153–159.
Cao, C.; Long, Y. T. Biological nanopores: Confined spaces for electrochemical single-molecule analysis. Acc. Chem. Res. 2018, 51, 331–341.
Lee, B.; Richards, F. M. The interpretation of protein structures: Estimation of static accessibility. J. Mol. Biol. 1971, 55, 379–400, IN3-IN4.
Honig, B.; Nicholls, A. Classical electrostatics in biology and chemistry. Science 1995, 268, 1144–1149.
Allen, M. P.; Tildesley, D. J. Computer Simulation of Liquids; Oxford University Press: Oxford, 2017.
Angelucci, F.; Miele, A. E.; Ardini, M.; Boumis, G.; Saccoccia, F.; Bellelli, A. Typical 2-Cys peroxiredoxins in human parasites: Several physiological roles for a potential chemotherapy target. Mol. Biochem. Parasitol. 2016, 206, 2–12.
Wang, H. Y.; Li, Y.; Qin, L. X.; Heyman, A.; Shoseyov, O.; Willner, I.; Long, Y. T.; Tian, H. Single-molecule DNA detection using a novel SP1 protein nanopore. Chem. Commun. 2013, 49, 1741–1743.
Ying, Y. L.; Cao, C.; Long, Y. T. Single molecule analysis by biological nanopore sensors. Analyst 2014, 139, 3826–3835.
Giovannini, G.; Ardini, M.; Maccaferri, N.; Zambrana-Puyalto, X.; Panella, G.; Angelucci, F.; Ippoliti, R.; Garoli, D.; De Angelis, F. Bioassisted tailored synthesis of plasmonic silver nanorings and site-selective deposition on graphene arrays. Adv. Opt. Mater. 2020, 8, 1901583.
Asandei, A.; Chinappi, M.; Lee, J. K.; Seo, C. H.; Mereuta, L.; Park, Y.; Luchian, T. Placement of oppositely charged aminoacids at a polypeptide termini determines the voltage-controlled braking of polymer transport through nanometer-scale pores. Sci. Rep. 2015, 5, 10419.
Restrepo-Pérez, L.; John, S.; Aksimentiev, A.; Joo, C.; Dekker, C. SDS-assisted protein transport through solid-state nanopores. Nanoscale 2017, 9, 11685–11693.
Cressiot, B.; Braselmann, E.; Oukhaled, A.; Elcock, A. H.; Pelta, J.; Clark, P. L. Dynamics and energy contributions for transport of unfolded pertactin through a protein nanopore. ACS Nano 2015, 9, 9050–9061.
Chinappi, M.; Luchian, T.; Cecconi, F. Nanopore tweezers: Voltage-controlled trapping and releasing of analytes. Phys. Rev. E 2015, 92, 032714.
Pastoriza-Gallego, M.; Breton, M. F.; Discala, F.; Auvray, L.; Betton, J. M.; Pelta, J. Evidence of unfolded protein translocation through a protein nanopore. ACS Nano 2014, 8, 11350–11360.
Kim, H. J.; Choi, U. J.; Kim, H.; Lee, K.; Park, K. B.; Kim, H. M.; Chi, S. W.; Lee, J. S.; Kim, K. B. Translocation of DNA and protein through a sequentially polymerized polyurea nanopore. Nanoscale 2019, 11, 444–453.
Mereuta, L.; Roy, M.; Asandei, A.; Lee, J. K.; Park, Y.; Andricioaei, I.; Luchian, T. Slowing down single-molecule trafficking through a protein nanopore reveals intermediates for peptide translocation. Sci. Rep. 2014, 4, 3885.
Plesa, C.; Kowalczyk, S. W.; Zinsmeester, R.; Grosberg, A. Y.; Rabin, Y.; Dekker, C. Fast translocation of proteins through solid state nanopores. Nano Lett. 2013, 13, 658–663.
Rodriguez-Larrea, D.; Bayley, H. Multistep protein unfolding during nanopore translocation. Nat. Nanotechnol. 2013, 8, 288–295.
Ayub, M.; Bayley, H. Engineered transmembrane pores. Curr. Opin. Chem. Biol. 2016, 34, 117–126.
Roy, R.; Hohng, S.; Ha, T. A practical guide to single-molecule FRET. Nat. Methods 2008, 5, 507–516.
Dimura, M.; Peulen, T. O.; Hanke, C. A.; Prakash, A.; Gohlke, H.; Seidel, C. A. M. Quantitative FRET studies and integrative modeling unravel the structure and dynamics of biomolecular systems. Curr. Opin. Struct. Biol. 2016, 40, 163–185.
Hoefling, M.; Grubmüller, H. In silico FRET from simulated dye dynamics. Comput. Phys. Commun. 2013, 184, 841–852.
Acknowledgements
The research leading to these results has received funding from the Horizon 2020 Program, FET-Open: PROSEQO, Grant Agreement no. [687089]. We acknowledge PRACE for awarding us access to Marconi at CINECA, Italy.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Funding note
Open Access funding provided by Istituto Italiano di Tecnologia within the CRUICARE Agreement.
Electronic Supplementary Material
Rights and permissions
This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Spitaleri, A., Garoli, D., Schütte, M. et al. Adaptive nanopores: A bioinspired label-free approach for protein sequencing and identification. Nano Res. 14, 328–333 (2021). https://doi.org/10.1007/s12274-020-3095-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12274-020-3095-z
Keywords
- nanopores
- single molecule sequencing
- protein sequencing
- luorescence resonance energy transfer (FRET)
- amino-acids
- fluorescence