Abstract
Post-translational modifications alter the properties of proteins through the cleavage of peptide bonds or the addition of a modifying group to one or more amino acids1,2,3,4,5,6,7. These modifications allow proteins to perform their primary biological functions8,9,10,11, but single-protein studies of post-translational modifications have been hindered by a lack of suitable analysis methods12,13,14,15,16. Here, we show that single amino acids can be identified using electron tunnelling currents measured as the individual molecules pass through a nanoscale gap between electrodes. We identify 12 different amino acids and the post-translational modification phosphotyrosine, which is involved in the process that switches enzymes on and off17,18,19,20. Furthermore, we show that the conductance measurements can be used to partially sequence peptides of an epidermal growth factor receptor substrate, and can discriminate a peptide from its phosphorylated variant.
Similar content being viewed by others
References
Pandey, A. & Mann, M. Proteomics to study genes and genomes. Nature 405, 837–846 (2000).
Mann, M. & Jensen, O. N. Proteomic analysis of post-translational modifications. Nature Biotechnol. 21, 255–261 (2003).
Steen, H. & Mann, M. The abc's (and xyz's) of peptide sequencing. Nature Rev. Mol. Cell Biol. 5, 699–711 (2004).
Tran, J. C. et al. Mapping intact protein isoforms in discovery mode using top-down proteomics. Nature 480, 254–258 (2011).
Cravatt, B. F., Simon, G. M. & Yates, J. R. The biological impact of mass-spectrometry-based proteomics. Nature 450, 991–1000 (2007).
Domon, B. & Aebersold, R. Review—mass spectrometry and protein analysis. Science 312, 212–217 (2006).
Seet, B. T., Dikic, I., Zhou, M. M. & Pawson, T. Reading protein modifications with interaction domains. Nature Rev. Mol. Cell Biol. 7, 473–483 (2006).
Choudhary, C. et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325, 834–840 (2009).
Vucic, D., Dixit, V. M. & Wertz, I. E. Ubiquitylation in apoptosis: a post-translational modification at the edge of life and death. Nature Rev. Mol. Cell Biol. 12, 439–452 (2011).
Kouzarides, T. Chromatin modifications and their function. Cell 128, 693–705 (2007).
Tsur, D., Tanner, S., Zandi, E., Bafna, V. & Pevzner, P. A. Identification of post-translational modifications by blind search of mass spectra. Nature Biotechnol. 23, 1562–1567 (2005).
Mertins, P. et al. Integrated proteomic analysis of post-translational modifications by serial enrichment. Nature Methods 10, 634–637 (2013).
Doerr, A. Mass spectrometry-based targeted proteomics. Nature Methods 10, 23–23 (2013).
Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003).
Zhu, H. et al. Analysis of yeast protein kinases using protein chips. Nature Genet. 26, 283–289 (2000).
Witze, E. S., Old, W. M., Resing, K. A. & Ahn, N. G. Mapping protein post-translational modifications with mass spectrometry. Nature Methods 4, 798–806 (2007).
Olsen, J. V. et al. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci. Signal 3, ra3 (2010).
Manning, G., Whyte, D. B., Martinez, R., Hunter, T. & Sudarsanam, S. The protein kinase complement of the human genome. Science 298, 1912–1934 (2002).
Oda, Y., Nagasu, T. & Chait, B. T. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nature Biotechnol. 19, 379–382 (2001).
Tran, J. C. et al. Mapping intact protein isoforms in discovery mode using top-down proteomics. Nature 480, 254–258 (2011).
Tsutsui, M., Shoji, K., Taniguchi, M. & Kawai, T. Formation and self-breaking mechanism of stable atom-sized junctions. Nano Lett. 8, 345–349 (2008).
Agrait, N., Yeyati, A. L. & van Ruitenbeek, J. M. Quantum properties of atomic-sized conductors. Phys. Rep. 377, 81–279 (2003).
Tsutsui, M., Taniguchi, M., Yokota, K. & Kawai, T. Identifying single nucleotides by tunnelling current. Nature Nanotech. 5, 286–290 (2010).
Ohshiro, T. et al. Single-molecule electrical random resequencing of DNA and RNA. Sci. Rep. 2, 501 (2012).
Taniguchi, M., Tsutsui, M., Shoji, K., Fujiwara, H. & Kawai, T. Single-molecule junctions with strong molecule–electrode coupling. J. Am. Chem. Soc. 131, 14146–14147 (2009).
Fernandez-Torrente, I. et al. Long-range repulsive interaction between molecules on a metal surface induced by charge transfer. Phys. Rev. Lett. 99, 176103 (2007).
Chen, F., Li, X., Hihath, J., Huang, Z. & Tao, N. Effect of anchoring groups on single-molecule conductance: comparative study of thiol-, amine-, and carboxylic-acid-terminated molecules. J. Am. Chem. Soc. 128, 15874–15881 (2006).
Zhao, Y. et al. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling. Nature Nanotech. 9, 466–473 (2014).
Noble, M. E. M., Endicott, J. A. & Johnson, L. N. Protein kinase inhibitors: insights into drug design from structure. Science 303, 1800–1805 (2004).
Sharma, S. V., Bell, D. W., Settleman, J. & Haber, D. A. Epidermal growth factor receptor mutations in lung cancer. Nature Rev. Cancer 7, 169–181 (2007).
Acknowledgements
This research was partially supported by the Japan Society for the Promotion of Science (JSPS) through its Funding Program for World-Leading Innovative R&D on Science and Technology, and KAKENHI grant no. 26220603.
Author information
Authors and Affiliations
Contributions
M.Ta. and T.K. planned and designed the experiments. T.O., M.Ts., K.Y. and M.F. participated in fabrications of nano-MCBJs and single-molecule detection measurements. T.O., M.Ts., K.Y. and M.Ta. performed data analyses. T.O., M.Ta. and T.K. co-wrote the paper.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary information
Supplementary Information (PDF 1500 kb)
Rights and permissions
About this article
Cite this article
Ohshiro, T., Tsutsui, M., Yokota, K. et al. Detection of post-translational modifications in single peptides using electron tunnelling currents. Nature Nanotech 9, 835–840 (2014). https://doi.org/10.1038/nnano.2014.193
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nnano.2014.193
- Springer Nature Limited
This article is cited by
-
Peptide sequencing based on host–guest interaction-assisted nanopore sensing
Nature Methods (2024)
-
MoS2 nanopore identifies single amino acids with sub-1 Dalton resolution
Nature Communications (2023)
-
Molecular sensitised probe for amino acid recognition within peptide sequences
Nature Communications (2023)
-
Direct biomolecule discrimination in mixed samples using nanogap-based single-molecule electrical measurement
Scientific Reports (2023)
-
Single-molecule mechanical fingerprinting with DNA nanoswitch calipers
Nature Nanotechnology (2021)