Paving the way to single-molecule protein sequencing

Abstract

Proteins are major building blocks of life. The protein content of a cell and an organism provides key information for the understanding of biological processes and disease. Despite the importance of protein analysis, only a handful of techniques are available to determine protein sequences, and these methods face limitations, for example, requiring a sizable amount of sample. Single-molecule techniques would revolutionize proteomics research, providing ultimate sensitivity for the detection of low-abundance proteins and the realization of single-cell proteomics. In recent years, novel single-molecule protein sequencing schemes that use fluorescence, tunnelling currents and nanopores have been proposed. Here, we present a review of these approaches, together with the first experimental efforts towards their realization. We discuss their advantages and drawbacks, and present our perspective on the development of single-molecule protein sequencing techniques.

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Fig. 1: Schematic of the single-molecule protein sequencing workflow with fluorescence, nanopores or tunnelling currents.
Fig. 2: Protein fingerprinting schemes using fluorescence.
Fig. 3: Amino acid and peptide characterization with tunnelling currents.
Fig. 4: Translocation of peptides and unfolded proteins through nanopores.
Fig. 5: Translocation of unfolded proteins through nanopores using an oligonucleotide linker (left) or an unfoldase (right).

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Acknowledgements

We thank S. Pud, S. Schmid, S. Caneva, J. van Ginkel and M. Filius for discussions. We acknowledge funding received from the Netherlands Organisation for Scientific Research (NWO/OCW) as a part of the Frontiers of Nanoscience programme. The C.D. lab was further supported by the ERC Advanced Grant SynDiv (No. 669598) and by the National Human Genome Research Institute of the National Institute of Health under Award Number R01-HG007406. C.J. was funded by the Foundation for Fundamental Research on Matter (12PR3029 and SMPS).

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Restrepo-Pérez, L., Joo, C. & Dekker, C. Paving the way to single-molecule protein sequencing. Nature Nanotech 13, 786–796 (2018). https://doi.org/10.1038/s41565-018-0236-6

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