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Single-molecule peptide sequencing through reverse translation of peptides into DNA

Nature Biotechnology (2026)Cite this article

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Abstract

Despite advances in mass spectrometry and emerging single-molecule approaches, sequencing peptides at the single-molecule level remains a central challenge in proteomics. Here we present a ‘reverse translation’ strategy that enables single-molecule peptide sequencing with single-amino-acid resolution. In this approach, peptides undergo a modified Edman degradation that iteratively releases N-terminal amino acids tagged with peptide-specific DNA barcodes. Antibody-mediated proximity extension assays identify these barcoded amino acids and generate PCR-amplifiable DNA reporters that record the identity, position and originating peptide of each amino acid. The resulting DNA library is directly read by high-throughput sequencing, converting peptide sequences into digital DNA outputs. Using this approach, we demonstrate true single-molecule peptide sequencing, achieving full sequence coverage in millions of reads and accurate differentiation of both native and post-translationally modified peptides. These results establish a framework that redefines protein sequencing as a DNA sequencing problem and lays the foundation for high-throughput, de novo single-molecule protein sequencing.

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Fig. 1: Overview of peptide sequencing through reverse translation.
Fig. 2: Developing DNA-encoded Edman degradation.
Fig. 3: PEA-mediated conversion of DNA-barcoded PTC amino acids into DNA reporters.
Fig. 4: Demonstrations of ensemble peptide sequencing.
Fig. 5: Single-molecule peptide sequencing.

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Data availability

The data supporting the findings of this study are available within the article and its Supplementary Information. The next-generation sequencing reads for ensemble and single-molecule peptide sequencing were deposited to the National Center for Biotechnology Information Sequence Read Archive under BioProjects PRJNA1420480 and PRJNA1423337, respectively. Source data are provided with this paper.

Code availability

The C++ and Python scripts for data processing and visualization of single-molecule peptide sequencing are available from GitHub (https://github.com/whulwzheng-source/smPeptideSeq).

Change history

  • 25 March 2026

    In the version of this article initially published, the Peer review information section was incomplete and is now amended in the HTML and PDF versions of the article.

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Acknowledgements

This work was supported by the Helmsley Trust, Wellcome LEAP SAVE program. We thank A. Hugenmatter at Stanford Innovative Medicines Accelerator for the helpful discussion. We thank J. Lowitz at Antibody Solutions for his assistance with custom antibody generation. We thank T. McLaughlin at Stanford University MS for her assistance with developing LC–MS methods for oligonucleotides. This work was supported by the Vincent Coates Foundation MS Laboratory, Stanford University MS (RRID:SCR_017801) using the Bruker Microflex MALDI-TOF MS instrument (RRID:SCR_018696) and Thermo Exploris 240 LC–MS system (RRID:SCR_022216) that was purchased with funding from Stanford C-ShaRP (RRID:SCR_022986).

Author information

Authors and Affiliations

  1. Department of Radiology, Stanford University, Stanford, CA, USA

    Liwei Zheng, Yujia Sun, Michael Eisenstein & Hyongsok Tom Soh

  2. Department of Electrical Engineering, Stanford University, Stanford, CA, USA

    Linus A. Hein, Michael Eisenstein & Hyongsok Tom Soh

  3. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Hyongsok Tom Soh

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  1. Liwei Zheng
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  2. Yujia Sun
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  3. Linus A. Hein
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Contributions

L.Z. and H.T.S. conceptualized the study. L.Z. and Y.S. performed the experiments and analyzed the experimental data. L.Z. and L.A.H. analyzed the single-molecule peptide sequencing data. H.T.S. supervised the research. L.Z. and H.T.S. wrote the original draft. L.Z., Y.S., M.E. and H.T.S. reviewed and edited the paper.

Corresponding authors

Correspondence to Liwei Zheng or Hyongsok Tom Soh.

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Competing interests

L.Z., Y.S. and H.T.S. are listed as coinventors on a pending patent application related to this work filed at the US Patent and Trademark Office (no. PCT/US2024/017167). L.A.H. and M.E. declare no competing interests.

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Nature Biotechnology thanks Chirlmin Joo, Stefan Howorka and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information (download PDF )

Materials, procedure for the preparation of oligonucleotides and organic compounds, Supplementary Text, Figs. 1–18 and Tables 1, 3–7, MS spectra of oligonucleotides, MS spectra of antibody–oligonucleotide conjugates, nuclear magnetic resonance spectra of organic compounds and uncropped gel images.

Reporting Summary (download PDF )

Supplementary Table 2 (download XLSX )

List of oligonucleotides.

Source data

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Zheng, L., Sun, Y., Hein, L.A. et al. Single-molecule peptide sequencing through reverse translation of peptides into DNA. Nat Biotechnol (2026). https://doi.org/10.1038/s41587-026-03061-z

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