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Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling

Nature Nanotechnology volume 9, pages 466–473 (2014)Cite this article

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Abstract

The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single-molecule protein sequencing is a critical step in the search for protein biomarkers. Here, we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules, then measuring the electron tunnelling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic ‘fingerprints’ associated with each binding motif. With this recognition tunnelling technique, we are able to identify D and L enantiomers, a methylated amino acid, isobaric isomers and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore.

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Figure 1: Recognition tunnelling (RT).
Figure 2: Examples of RT signals from amino acids.
Figure 3: Signal features identify analytes.
Figure 4: Closely related pairs of analytes can be significantly separated (>80%) using just two signal features together.
Figure 5: A mixture produces alternating cluster signals as different molecules diffuse into and out of the gap.

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Acknowledgements

S. Chang assisted in the original survey of amino acids. The authors thank P. Pang, P. Krstic, C. Hernandez-Suarez and W. Offenberg for useful discussions. This work was supported in part by a DNA sequencing technology grant from the NHGRI (HG 006323).

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Author notes

  1. Yanan Zhao and Brian Ashcroft: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Physics, Arizona State University, PO Box 871504, Tempe, 85287, Arizona, USA

    Yanan Zhao, JongOne Im & Stuart Lindsay

  2. Biodesign Institute, Arizona State University, PO Box 875001, Tempe, 85287, Arizona, USA

    Yanan Zhao, Brian Ashcroft, Peiming Zhang, Hao Liu, Suman Sen, Weisi Song, JongOne Im, Brett Gyarfas, Saikat Manna, Sovan Biswas, Chad Borges & Stuart Lindsay

  3. Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, 85287, Arizona, USA

    Hao Liu, Suman Sen, Weisi Song, Saikat Manna, Sovan Biswas, Chad Borges & Stuart Lindsay

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  1. Yanan Zhao
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Contributions

Y.Z. and H.L. carried out tunnelling measurements with assistance from S.S., W.S. and J.I., B.A. wrote the SVM code and analysed data. B.G. contributed to the analysis. S.M. carried out force spectroscopy experiments. C.B. and S.B. carried out the electrospray MS., P.Z. and S.L. designed experiments and S.L. wrote the paper.

Corresponding author

Correspondence to Stuart Lindsay.

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Competing interests

Y.Z., P.Z. and S.L. are named as inventors in a patent application (patent application number PCT/US13/24,130; title: Device for reading amino acid sequence inventors). S.L. is co-founder of a company that has a licence option on the technology.

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Zhao, Y., Ashcroft, B., Zhang, P. et al. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling. Nature Nanotech 9, 466–473 (2014). https://doi.org/10.1038/nnano.2014.54

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