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

Nature Nanotechnology volume 11, pages 968–976 (2016)Cite this article

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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 nm3, but it is not sensitive enough to discriminate between the volumes of all twenty amino acids.

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Figure 1: Detecting single proteins using a subnanopore.
Figure 2: Detecting amino acids in a single protein using a subnanopore.
Figure 3: Protein sequence analysis.
Figure 4: Testing the sensitivity of a subnanopore to molecular volume.

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Acknowledgements

This work was partially supported by a grant from the National Science Foundation (DBI 1256052), the Keough–Hesburgh Professorship and the Walther Cancer Foundation. We gratefully acknowledge conversations with J. Barthel and critical readings of the manuscript by W. Timp and T. Tanaka.

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Author notes

  1. Eamonn Kennedy and Zhuxin Dong: These authors contributed equally to this work

Authors and Affiliations

  1. Electrical Engineering, University of Notre Dame, Notre Dame, 46556, Indiana, USA

    Eamonn Kennedy & Zhuxin Dong

  2. Chemical Engineering, University of Notre Dame, Notre Dame, 46556, Indiana, USA

    Clare Tennant

  3. Electrical Engineering and Biological Science, University of Notre Dame, Notre Dame, 46556, Indiana, USA

    Gregory Timp

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  1. Eamonn Kennedy
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  2. Zhuxin Dong
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Contributions

G.T. conceived the experiments. E.K. and Z.D. designed the experiments in consultation with G.T. E.K., Z.D. and C.T. performed the experiments and analysed the data. E.K. developed the algorithms used to analyse the current blockade data, in particular. G.T., E.K. and Z.D. co-wrote the manuscript, but all of the authors discussed the results and commented on it.

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Correspondence to Gregory Timp.

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Kennedy, E., Dong, Z., Tennant, C. et al. Reading the primary structure of a protein with 0.07 nm3 resolution using a subnanometre-diameter pore. Nature Nanotech 11, 968–976 (2016). https://doi.org/10.1038/nnano.2016.120

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