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Full-length single-molecule protein fingerprinting

Nature Nanotechnology (2024)Cite this article

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Abstract

Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O-GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis.

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Fig. 1: Repetitive binding of short DNA imager strands allows for high-resolution protein fingerprinting.
Fig. 2: Single-molecule protein fingerprinting of disordered and folded proteins.
Fig. 3: PTM mapping using FRET X.
Fig. 4: Single-molecule protein fingerprinting using N-terminal labelling.

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Data availability

The data supporting the main finding of this study are available at the publicly accessible online repository Zenodo with identifier https://doi.org/10.5281/zenodo.10179066. Any additional data are available from the corresponding author upon request.

Code availability

The algorithms for the codes supporting the main findings of this study are available at https://doi.org/10.5281/zenodo.10156504. Any additional information concerning the code is available from the corresponding author upon request.

References

  1. Aebersold, R. et al. How many human proteoforms are there? Nat. Chem. Biol. 14, 206–214 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kim, H. K., Pham, M. H. C., Ko, K. S., Rhee, B. D. & Han, J. Alternative splicing isoforms in health and disease. Pflügers Arch. 470, 995–1016 (2018).

  3. Paronetto, M. P., Passacantilli, I. & Sette, C. Alternative splicing and cell survival: from tissue homeostasis to disease. Cell Death Differ. 23, 1919–1929 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lin, H. & Caroll, K. S. Introduction: posttranslational protein modification. Chem. Rev. 118, 887–888 (2018).

    Article  CAS  PubMed  Google Scholar 

  5. Carbonara, K., Andonovski, M. & Coorssen, J. R. Proteomes are of proteoforms: embracing the complexity. Proteomes 9, 38 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Benson, M. D., Ngo, D., Ganz, P. & Gerszten, R. E. Emerging affinity reagents for high throughput proteomics: trust, but verify. Circulation 140, 1610–1612 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Yang, Y. et al. Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity. Nat. Commun. 7, 13397 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Čaval, T., Tian, W., Yang, Z., Clausen, H. & Heck, A. J. R. Direct quality control of glycoengineered erythropoietin variants. Nat. Commun. 9, 3342 (2018).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  9. Siuti, N. & Kelleher, N. L. Decoding protein modifications using top-down mass spectrometry. Nat. Methods 410, 817–821 (2007).

    Article  Google Scholar 

  10. Wang, Y., Zhao, Y., Bollas, A., Wang, Y. & Au, K. F. Nanopore sequencing technology, bioinformatics and applications. Nat. Biotechnol. 39, 1348–1365 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ardui, S., Ameur, A., Vermeesch, J. R. & Hestand, M. S. Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics. Nucleic Acids Res. 46, 2159–2168 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Restrepo-Pérez, L., Joo, C. & Dekker, C. Paving the way to single-molecule protein sequencing. Nat. Nanotechnol. 13, 786–796 (2018).

    Article  ADS  PubMed  Google Scholar 

  13. Alfaro, J. A. et al. The emerging landscape of single-molecule protein sequencing technologies. Nat. Methods 18, 604–617 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Floyd, B. M. & Marcotte, E. M. Protein sequencing, one molecule at a time. Annu. Rev. Biophys. 51, 181–200 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Timp, W. & Timp, G. Beyond mass spectrometry, the next step in proteomics. Sci. Adv. 6, eaax8978 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  16. Swaminathan, J., Boulgakov, A. A. & Marcotte, E. M. A theoretical justification for single molecule peptide sequencing. PLoS Comput. Biol. 11, e1004080 (2015).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  17. Rodriques, S. G., Marblestone, A. H. & Boyden, E. S. A theoretical analysis of single molecule protein sequencing via weak binding spectra. PLoS ONE 14, e0212868 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yao, Y., Docter, M., Van Ginkel, J., De Ridder, D. & Joo, C. Single-molecule protein sequencing through fingerprinting: computational assessment. Phys. Biol. 12, 10–16 (2015).

    Article  Google Scholar 

  19. de Lannoy, C. V. et al. Evaluation of FRET X for single-molecule protein fingerprinting. iScience 24, 103239 (2021).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  20. Yu, L. et al. Unidirectional single-file transport of full-length proteins through a nanopore. Nat. Biotechnol. 41, 1130–1139 (2023).

  21. van Ginkel, J. et al. Single-molecule peptide fingerprinting. Proc. Natl Acad. Sci. USA 115, 3338–3343 (2018).

  22. Swaminathan, J. et al. Highly parallel single-molecule identification of proteins in zeptomole-scale mixtures. Nat. Biotechnol. 36, 1076–1082 (2018).

    Article  CAS  Google Scholar 

  23. Shrestha, P. et al. Single-molecule mechanical fingerprinting with DNA nanoswitch calipers. Nat. Nanotechnol. 16, 1362–1370 (2021).

  24. Filius, M., Kim, S. H., Severins, I. & Joo, C. High-resolution single-molecule FRET via DNA exchange (FRET X). Nano Lett. 21, 3295–3301 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. Filius, M., van Wee, R. & Joo, C. in Single Molecule Analysis: Methods and Protocols (eds Heller, I. et al.) 203–213 (Springer, 2024).

  26. Van Wee, R., Filius, M. & Joo, C. Completing the canvas: advances and challenges for DNA-PAINT super-resolution imaging. Trends Biochem. Sci. 11, 918–930 (2021).

    Google Scholar 

  27. Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Super-resolution microscopy with DNA-PAINT. Nat. Protoc. 12, 1198–1228 (2017).

    Article  CAS  PubMed  Google Scholar 

  28. Shi, X. et al. Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging. Nat. Methods 9, 499–503 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Schuler, B. & Hofmann, H. Single-molecule spectroscopy of protein folding dynamics—expanding scope and timescales. Curr. Opin. Struct. Biol. 23, 36–47 (2013).

    Article  CAS  PubMed  Google Scholar 

  30. Yang, X. & Qian, K. Protein O-GlcNAcylation: emerging mechanisms and functions. Nat. Rev. Mol. Cell Biol. 18, 452–465 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Vellosillo, P. & Minguez, P. A global map of associations between types of protein posttranslational modifications and human genetic diseases. iScience 24, 102917 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Mauri, T. et al. O-GlcNAcylation prediction: an unattained objective. Adv. Appl. Bioinform. Chem. 14, 87–102 (2021).

  33. Shi, J., Ruijtenbeek, R. & Pieters, R. J. Demystifying O-GlcNAcylation: hints from peptide substrates. Glycobiology 28, 814–824 (2018).

    Article  CAS  PubMed  Google Scholar 

  34. Shen, D. L. et al. Catalytic promiscuity of O-GlcNAc transferase enables unexpected metabolic engineering of cytoplasmic proteins with 2-azido-2-deoxy-glucose. ACS Chem. Biol. 12, 206–213 (2017).

    Article  CAS  PubMed  Google Scholar 

  35. Mayer, A., Gloster, T. M., Chou, W. K., Vocadlo, D. J. & Tanner, M. E. 6′-Azido-6′-deoxy-UDP-N-acetylglucosamine as a glycosyltransferase substrate. Bioorg. Med. Chem. Lett. 21, 1199–1201 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Macdonald, J. I., Munch, H. K., Moore, T. & Francis, M. B. One-step site-specific modification of native proteins with 2-pyridinecarboxyaldehydes. Nat. Chem. Biol. 11, 326–331 (2015).

    Article  CAS  PubMed  Google Scholar 

  37. Wang, S. et al. S100A8/A9 in inflammation. Front. Immunol. 9, 1298 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Vijayan, A. L. et al. Procalcitonin: a promising diagnostic marker for sepsis and antibiotic therapy. J. Intensive Care 5, 51 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Senior, A. W. et al. Improved protein structure prediction using potentials from deep learning. Nature 577, 706–710 (2020).

    Article  ADS  CAS  PubMed  Google Scholar 

  40. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  41. Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313–318 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Erickson, H. P. Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy. Biol. Proced. Online 11, 32–51 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Ree, R., Varland, S. & Arnesen, T. Spotlight on protein N-terminal acetylation. Exp. Mol. Med. 50, 1–13 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Bloom, S. et al. Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials. Nat. Chem. 10, 205–211 (2018).

    Article  CAS  PubMed  Google Scholar 

  45. Ramirez, D. H. et al. Engineering a proximity-directed O-GlcNAc transferase for selective protein O-GlcNAcylation in cells. ACS Chem. Biol. 15, 1059–1066 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Yang, Y.-Y., Ascano, J. M. & Hang, H. C. Bioorthogonal chemical reporters for monitoring protein acetylation. J. Am. Chem. Soc. 132, 3640–3641 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Westcott, N. P., Fernandez, J. P., Molina, H. & Hang, H. C. Chemical proteomics reveals ADP-ribosylation of small GTPases during oxidative stress. Nat. Chem. Biol. 13, 302–308 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Rabuka, D., Hubbard, S. C., Laughlin, S. T., Argade, S. P. & Bertozzi, C. R. A chemical reporter strategy to probe glycoprotein fucosylation. J. Am. Chem. Soc. 128, 12078–12079 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Boeggeman, E. et al. Direct identification of nonreducing GlcNAc residues on N-glycans of glycoproteins using a novel chemoenzymatic method. Bioconjugate Chem. 18, 806–814 (2007).

    Article  CAS  Google Scholar 

  50. van Geel, R. et al. Chemoenzymatic conjugation of toxic payloads to the globally conserved N-glycan of native mAbs provides homogeneous and highly efficacious antibody–drug conjugates. Bioconjugate Chem. 26, 2233–2242 (2015).

    Article  Google Scholar 

  51. Tate, E. W., Kalesh, K. A., Lanyon-Hogg, T., Storck, E. M. & Thinon, E. Global profiling of protein lipidation using chemical proteomic technologies. Curr. Opin. Chem. Biol. 24, 48–57 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Anderson, N. L. & Anderson, N. G. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteom. 1, 845–867 (2002).

    Article  CAS  Google Scholar 

  53. Han, X., Aslanian, A. & Yates, J. R. Mass spectrometry for proteomics. Curr. Opin. Chem. Biol. 12, 483–490 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Filius, M. et al. High-speed super-resolution imaging using protein-assisted DNA-PAINT. Nano Lett. 20, 2264–2270 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  55. Kim, S. H., Kim, H., Jeong, H. & Yoon, T. Y. Encoding multiple virtual signals in DNA barcodes with single-molecule FRET. Nano Lett. 21, 1694–1701 (2021).

    Article  ADS  CAS  PubMed  Google Scholar 

  56. McCann, J. J., Choi, U. B., Zheng, L., Weninger, K. & Bowen, M. E. Optimizing methods to recover absolute FRET efficiency from immobilized single molecules. Biophys. J. 99, 961–970 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  57. Cristianini, N. & Shawe-Taylor, J. An Introduction to Support Vector Machines and Other Kernel-based Learning Methods (Cambridge University Press, 2000).

  58. Pedregosa, F. et al. Scikit-learn: machine learning in Python. J. Mach. Learn. Res. 12, 2825–2830 (2011).

    MathSciNet  Google Scholar 

  59. Pabst, M. et al. A general approach to explore prokaryotic protein glycosylation reveals the unique surface layer modulation of an anammox bacterium. ISME J. 16, 346–357 (2022).

    Article  CAS  PubMed  Google Scholar 

  60. Chuh, K. N., Zaro, B. W., Piller, F., Piller, V. & Pratt, M. R. Changes in metabolic chemical reporter structure yield a selective probe of O-GlcNAc modification. J. Am. Chem. Soc. 136, 12283–12295 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

C.J. and D.d.R. acknowledge funding from NWO-I (SMPS). C.J. acknowledges funding from NWO (Vici, VI.C.202.015), the Basic Science Research Program (NRF-2023R1A2C2004745) and Frontier 10-10 (Ewha Womans University). Y.W. was funded by a Chinese Scholarship Council (CSC) grant.

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Author notes

  1. These authors contributed equally: Raman van Wee, Carlos de Lannoy.

Authors and Affiliations

  1. Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands

    Mike Filius, Raman van Wee, Carlos de Lannoy, Ilja Westerlaken, Zeshi Li, Sung Hyun Kim, Cecilia de Agrela Pinto & Chirlmin Joo

  2. Bioinformatics Group, Wageningen University, Wageningen, The Netherlands

    Carlos de Lannoy & Dick de Ridder

  3. Department of Physics, Ewha Womans University, Seoul, Republic of Korea

    Sung Hyun Kim & Chirlmin Joo

  4. Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

    Yunfei Wu & Geert-Jan Boons

  5. Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands

    Geert-Jan Boons

  6. Department of Biotechnology, Delft University of Technology, Delft, The Netherlands

    Martin Pabst

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Contributions

M.F. and C.J. initiated and designed the project. M.F. designed and performed the protein labelling procedures. M.F. and R.v.W. performed the single-molecule FRET X experiments. I.W. and C.d.A.P. expressed and purified the proteins. C.d.L. wrote the software for and performed the fingerprinting predictions and protein classification. D.d.R. supervised the fingerprinting prediction simulations. S.H.K. wrote the automated peak-finding code for single-molecule FRET X analysis. M.F. and Z.L. conceptualized the O-GlcNAc site mapping, and designed the chemoenzymatic and N-terminal labelling strategies. Y.W. and G.-J.B. synthesized UDP-azido-O-GlcNAc for protein O-GlcNAcylation. M.P. performed proteomic analysis. M.F., S.H.K., C.d.L. and C.J. analysed and discussed the data. M.F., R.v.W. and C.J. prepared the initial draft of the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Chirlmin Joo.

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Competing interests

C.J., M.F., C.d.L. and D.d.R. hold a patent on single-molecule FRET for protein characterization (patent number WO2021049940). C.J., M.F. and Z.L. have filed a patent for the bifunctional linker for N-terminal protein modification. The other authors declare no competing interests.

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Filius, M., van Wee, R., de Lannoy, C. et al. Full-length single-molecule protein fingerprinting. Nat. Nanotechnol. (2024). https://doi.org/10.1038/s41565-023-01598-7

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