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).
Wang, N. et al. Single-cell microRNA-mRNA co-sequencing reveals non-genetic heterogeneity and mechanisms of microRNA regulation. Nat. Commun. 10, 95 (2019).
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).
Sonneveld, S., Verhagen, B. M. P. & Tanenbaum, M. E. Heterogeneity in mRNA Translation. Trends Cell Biol. 30, 606–618 (2020).
Smith, L. M. et al. The Human Proteoform Project: defining the human proteome. Sci. Adv. 7, eabk0734 (2021).
Tajik, M., Baharfar, M. & Donald, W. A. Single-cell mass spectrometry. Trends Biotechnol. 40, 1374–1392 (2022).
Ye, Z. et al. One-Tip enables comprehensive proteome coverage in minimal cells and single zygotes. Nat. Commun. 15, 2474 (2024).
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).
MacCoss, M. J. et al. Sampling the proteome by emerging single-molecule and mass spectrometry methods. Nat. Methods 20, 339–346 (2023).
Alfaro, J. A. et al. The emerging landscape of single-molecule protein sequencing technologies. Nat. Methods 18, 604–617 (2021).
Restrepo-Pérez, L., Joo, C. & Dekker, C. Paving the way to single-molecule protein sequencing. Nat. Nanotechnol. 13, 786–796 (2018).
Reed, B. D. et al. Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device. Science 378, 186–192 (2022).
Martin-Baniandres, P. et al. Enzyme-less nanopore detection of post-translational modifications within long polypeptides. Nat. Nanotechnol. 18, 1335–1340 (2023).
Yu, L. et al. Unidirectional single-file transport of full-length proteins through a nanopore. Nat. Biotechnol. 41, 1130–1139 (2023).
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).
Nova, I. C. et al. Detection of phosphorylation post-translational modifications along single peptides with nanopores. Nat. Biotechnol. 42, 710–714 (2023).
Motone, K. et al. Multi-pass, single-molecule nanopore reading of long protein strands. Nature 633, 662–669 (2024).
Ouldali, H. et al. Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore. Nat. Biotechnol. 38, 176–181 (2020).
Wang, K. et al. Unambiguous discrimination of all 20 proteinogenic amino acids and their modifications by nanopore. Nat. Methods 21, 92–101 (2024).
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).
Swaminathan, J. et al. Highly parallel single-molecule identification of proteins in zeptomole-scale mixtures. Nat. Biotechnol. 36, 1076–1082 (2018).
Boutureira, O. & Bernardes, G. J. L. Advances in chemical protein modification. Chem. Rev. 115, 2174–2195 (2015).
Szijj, P. A., Kostadinova, K. A., Spears, R. J. & Chudasama, V. Tyrosine bioconjugation—an emergent alternative. Org. Biomol. Chem. 18, 9018–9028 (2020).
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).
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).
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).
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).
Zhang, P. et al. Highly sensitive serum protein analysis using magnetic bead-based proximity extension assay. Anal. Chem. 94, 12481–12489 (2022).
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).
Hong, J. M. et al. ProtSeq: toward high-throughput, single-molecule protein sequencing via amino acid conversion into DNA barcodes. iScience 25, 103586 (2022).
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).
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).
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).
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).
Potowski, M. et al. Chemically stabilized DNA barcodes for DNA-encoded chemistry. Angew. Chem. Int. Ed. 60, 19744–19749 (2021).
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).
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).
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).
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).
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).
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).
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).
Niall, H. D. Automated Edman degradation: the protein sequenator. Methods Enzymol. 27, 942–1010 (1973).
Laursen, R. A. Solid-phase Edman degradation. Eur. J. Biochem. 20, 89–102 (1971).
Fredriksson, S. et al. Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol. 20, 473–477 (2002).
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).
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).
Zhang, L. et al. Photoredox-catalyzed decarboxylative C-terminal differentiation for bulk- and single-molecule proteomics. ACS Chem. Biol. 16, 2595–2603 (2021).
Rehm, F. B. H. et al. Enzymatic C-terminal protein engineering with amines. J. Am. Chem. Soc. 143, 19498–19504 (2021).
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).
Boga, S. B. et al. Site-selective synthesis of insulin azides and bioconjugates. Bioconjug. Chem. 30, 1127–1132 (2019).
Edman, P. & Begg, G. A protein sequenator. Eur. J. Biochem. 1, 80–91 (1967).
Swaminathan, J., Boulgakov, A. A. & Marcotte, E. M. A theoretical justification for single molecule peptide sequencing. PLoS Comput. Biol. 11, e1004080 (2015).
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).
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).
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).
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).
— Source: Nature Biotechnology (https://www.nature.com/articles/s41587-026-03061-z)