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

Pollen, A. A. et al. Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex. Nat. Biotechnol. 32, 1053–1058 (2014).
Google Scholar
Wang, N. et al. Single-cell microRNA-mRNA co-sequencing reveals non-genetic heterogeneity and mechanisms of microRNA regulation. Nat. Commun. 10, 95 (2019).
Google Scholar
Kolodziejczyk, A. A., Kim, J. K., Svensson, V., Marioni, J. C. & Teichmann, S. A. The technology and biology of single-cell RNA sequencing. Mol. Cell 58, 610–620 (2015).
Google Scholar
Sonneveld, S., Verhagen, B. M. P. & Tanenbaum, M. E. Heterogeneity in mRNA Translation. Trends Cell Biol. 30, 606–618 (2020).
Google Scholar
Smith, L. M. et al. The Human Proteoform Project: defining the human proteome. Sci. Adv. 7, eabk0734 (2021).
Google Scholar
Tajik, M., Baharfar, M. & Donald, W. A. Single-cell mass spectrometry. Trends Biotechnol. 40, 1374–1392 (2022).
Google Scholar
Ye, Z. et al. One-Tip enables comprehensive proteome coverage in minimal cells and single zygotes. Nat. Commun. 15, 2474 (2024).
Google Scholar
Guzman, U. H. et al. Ultra-fast label-free quantification and comprehensive proteome coverage with narrow-window data-independent acquisition. Nat. Biotechnol. 1–12 https://doi.org/10.1038/s41587-023-02099-7 (2024).
Huffman, R. G. et al. Prioritized mass spectrometry increases the depth, sensitivity and data completeness of single-cell proteomics. Nat. Methods 20, 714–722 (2023).
Google Scholar
MacCoss, M. J. et al. Sampling the proteome by emerging single-molecule and mass spectrometry methods. Nat. Methods 20, 339–346 (2023).
Google Scholar
Alfaro, J. A. et al. The emerging landscape of single-molecule protein sequencing technologies. Nat. Methods 18, 604–617 (2021).
Google Scholar
Restrepo-Pérez, L., Joo, C. & Dekker, C. Paving the way to single-molecule protein sequencing. Nat. Nanotechnol. 13, 786–796 (2018).
Google Scholar
Reed, B. D. et al. Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device. Science 378, 186–192 (2022).
Google Scholar
Martin-Baniandres, P. et al. Enzyme-less nanopore detection of post-translational modifications within long polypeptides. Nat. Nanotechnol. 18, 1335–1340 (2023).
Google Scholar
Yu, L. et al. Unidirectional single-file transport of full-length proteins through a nanopore. Nat. Biotechnol. 41, 1130–1139 (2023).
Google Scholar
Brinkerhoff, H., Kang, A. S. W., Liu, J., Aksimentiev, A. & Dekker, C. Multiple rereads of single proteins at single-amino acid resolution using nanopores. Science 374, 1509–1513 (2021).
Google Scholar
Nova, I. C. et al. Detection of phosphorylation post-translational modifications along single peptides with nanopores. Nat. Biotechnol. 42, 710–714 (2023).
Google Scholar
Motone, K. et al. Multi-pass, single-molecule nanopore reading of long protein strands. Nature 633, 662–669 (2024).
Google Scholar
Ouldali, H. et al. Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore. Nat. Biotechnol. 38, 176–181 (2020).
Google Scholar
Wang, K. et al. Unambiguous discrimination of all 20 proteinogenic amino acids and their modifications by nanopore. Nat. Methods 21, 92–101 (2024).
Google Scholar
Zhang, M. et al. Real-time detection of 20 amino acids and discrimination of pathologically relevant peptides with functionalized nanopore. Nat. Methods 21, 609–618 (2024).
Google Scholar
Swaminathan, J. et al. Highly parallel single-molecule identification of proteins in zeptomole-scale mixtures. Nat. Biotechnol. 36, 1076–1082 (2018).
Google Scholar
Boutureira, O. & Bernardes, G. J. L. Advances in chemical protein modification. Chem. Rev. 115, 2174–2195 (2015).
Google Scholar
Szijj, P. A., Kostadinova, K. A., Spears, R. J. & Chudasama, V. Tyrosine bioconjugation—an emergent alternative. Org. Biomol. Chem. 18, 9018–9028 (2020).
Google Scholar
Choi, W. S. et al. Structural basis for the recognition of N-end rule substrates by the UBR box of ubiquitin ligases. Nat. Struct. Mol. Biol. 17, 1175–1181 (2010).
Google Scholar
Stein, B. J., Grant, R. A., Sauer, R. T. & Baker, T. A. Structural basis of an N-degron adaptor with more stringent specificity. Structure 24, 232–242 (2016).
Google Scholar
Edman, P., Högfeldt, E., Sillén, L. G. & Kinell, P.-O. Method for determination of the amino acid sequence in peptides. Acta Chem. Scand. 4, 283–293 (1950).
Google Scholar
McGregor, L. M., Gorin, D. J., Dumelin, C. E. & Liu, D. R. Interaction-dependent PCR: identification of ligand−target pairs from libraries of ligands and libraries of targets in a single solution-phase experiment. J. Am. Chem. Soc. 132, 15522–15524 (2010).
Google Scholar
Zhang, P. et al. Highly sensitive serum protein analysis using magnetic bead-based proximity extension assay. Anal. Chem. 94, 12481–12489 (2022).
Google Scholar
Zoltewicz, J. A., Clark, D. F., Sharpless, T. W. & Grahe, G. Kinetics and mechanism of the acid-catalyzed hydrolysis of some purine nucleosides. J. Am. Chem. Soc. 92, 1741–1750 (1970).
Google Scholar
Hong, J. M. et al. ProtSeq: toward high-throughput, single-molecule protein sequencing via amino acid conversion into DNA barcodes. iScience 25, 103586 (2022).
Google Scholar
Iida, T., Santa, T., Toriba, A. & Imai, K. Semi-automatic amino acid sequencing and D/L-configuration determination of peptides with detection of liberated N-terminal phenylthiocarbamoylamino acids. Analyst 123, 2829–2834 (1998).
Google Scholar
Matsunaga, H. et al. Proton: a major factor for the racemization and the dehydration at the cyclization/cleavage stage in the Edman sequencing method. Anal. Chem. 68, 2850–2856 (1996).
Google Scholar
Mitchell, M. J., Hirschowitz, W., Rastinejad, F. & Lu, P. Boron trifluoride–methanol complex as a non-depurinating detritylating agent in DNA synthesis. Nucleic Acids Res. 18, 5321 (1990).
Google Scholar
Seela, F., Menkhoff, S. & Behrendt, S. Furanoside–pyranoside isomerization of tubercidin and its 2′-deoxy derivatives: influence of nucleobase and sugar structure on the proton-catalysed reaction. J. Chem. Soc. Perkin Trans. 2, 525–530 (1986).
Google Scholar
Potowski, M. et al. Chemically stabilized DNA barcodes for DNA-encoded chemistry. Angew. Chem. Int. Ed. 60, 19744–19749 (2021).
Google Scholar
Farnsworth, V. & Steinberg, K. The generation of phenylthiocarbamyl or anilinothiazolinone amino acids from the postcleavage products of the Edman degradation. Anal. Biochem. 215, 200–210 (1993).
Google Scholar
Matsudaira, P. (ed.) A Practical Guide to Protein and Peptide Purification for Microsequencing (Academic Press, 1993).
Rydberg, P., Lüning, B., Wachtmeister, C. A., Eriksson, L. & Törnqvist, M. Applicability of a modified Edman procedure for measurement of protein adducts: mechanisms of formation and degradation of phenylthiohydantoins. Chem. Res. Toxicol. 15, 570–581 (2002).
Google Scholar
Flood, D. T. et al. Expanding reactivity in DNA-encoded library synthesis via reversible binding of DNA to an inert quaternary ammonium support. J. Am. Chem. Soc. 141, 9998–10006 (2019).
Google Scholar
MacConnell, A. B., McEnaney, P. J., Cavett, V. J. & Paegel, B. M. DNA-encoded solid-phase synthesis: encoding language design and complex oligomer library synthesis. ACS Comb. Sci. 17, 518–534 (2015).
Google Scholar
Halpin, D. R., Lee, J. A., Wrenn, S. J. & Harbury, P. B. DNA display III. Solid-phase organic synthesis on unprotected DNA. PLoS Biol. 2, e175 (2004).
Google Scholar
Wang, Z., Li, D., Tian, X. & Zhang, C. A copper-free and enzyme-free click chemistry-mediated single quantum dot nanosensor for accurate detection of microRNAs in cancer cells and tissues. Chem. Sci. 12, 10426–10435 (2021).
Google Scholar
Nakano, S. & Sugimoto, N. The structural stability and catalytic activity of DNA and RNA oligonucleotides in the presence of organic solvents. Biophys. Rev. 8, 11–23 (2016).
Google Scholar
Niall, H. D. Automated Edman degradation: the protein sequenator. Methods Enzymol. 27, 942–1010 (1973).
Google Scholar
Laursen, R. A. Solid-phase Edman degradation. Eur. J. Biochem. 20, 89–102 (1971).
Google Scholar
Fredriksson, S. et al. Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol. 20, 473–477 (2002).
Google Scholar
Budnik, B., Levy, E., Harmange, G. & Slavov, N. SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation. Genome Biol. 19, 161 (2018).
Google Scholar
Kokoris, M. et al. Sequencing by expansion (SBX)—a novel, high-throughput single-molecule sequencing technology. Preprint at bioRxiv https://doi.org/10.1101/2025.02.19.639056 (2025).
Bloom, S. et al. Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials. Nat. Chem. 10, 205–211 (2018).
Google Scholar
Zhang, L. et al. Photoredox-catalyzed decarboxylative C-terminal differentiation for bulk- and single-molecule proteomics. ACS Chem. Biol. 16, 2595–2603 (2021).
Google Scholar
Rehm, F. B. H. et al. Enzymatic C-terminal protein engineering with amines. J. Am. Chem. Soc. 143, 19498–19504 (2021).
Google Scholar
Antos, J. M. et al. Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity. J. Am. Chem. Soc. 131, 10800–10801 (2009).
Google Scholar
Boga, S. B. et al. Site-selective synthesis of insulin azides and bioconjugates. Bioconjug. Chem. 30, 1127–1132 (2019).
Google Scholar
Edman, P. & Begg, G. A protein sequenator. Eur. J. Biochem. 1, 80–91 (1967).
Google Scholar
Swaminathan, J., Boulgakov, A. A. & Marcotte, E. M. A theoretical justification for single molecule peptide sequencing. PLoS Comput. Biol. 11, e1004080 (2015).
Google Scholar
Yang, K.-A. et al. Recognition and sensing of low-epitope targets via ternary complexes with oligonucleotides and synthetic receptors. Nat. Chem. 6, 1003–1008 (2014).
Google Scholar
Boder, E. T., Midelfort, K. S. & Wittrup, K. D. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc. Natl Acad. Sci. USA 97, 10701–10705 (2000).
Google Scholar
Javanpour, A. A. & Liu, C. C. Evolving small-molecule biosensors with improved performance and reprogrammed ligand preference using OrthoRep. ACS Synth. Biol. 10, 2705–2714 (2021).
Google Scholar
Lee, G. R. et al. Small-molecule binding and sensing with a designed protein family. Preprint at bioRxiv https://doi.org/10.1101/2023.11.01.565201 (2023).
An, L. et al. Binding and sensing diverse small molecules using shape-complementary pseudocycles. Science 385, 276–282 (2024).
Google Scholar


