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Detection of phosphorylation post-translational modifications along single peptides with nanopores

Abstract

Current methods to detect post-translational modifications of proteins, such as phosphate groups, cannot measure single molecules or differentiate between closely spaced phosphorylation sites. We detect post-translational modifications at the single-molecule level on immunopeptide sequences with cancer-associated phosphate variants by controllably drawing the peptide through the sensing region of a nanopore. We discriminate peptide sequences with one or two closely spaced phosphates with 95% accuracy for individual reads of single molecules.

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Fig. 1: Nanopore PTM detection experimental schematic and data workflow.
Fig. 2: MCMC calculations of phosphate-containing peptides.

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Data availability

The raw nanopore data for all of the reads used in this study are publicly available at https://doi.org/10.57760/sciencedb.0833831.

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Acknowledgements

We thank A. Laszlo for discussions on the MCMC calculations, J. van der Torre for help in troubleshooting POC construction, E. van der Sluis and A. Goutou for Hel308 purification, and A. Aksimentiev for discussions. The work was supported by funding from the Dutch Research Council (NWO) project NWO-I680 (SMPS) (C.D.); European Research Council Advanced Grant 883684 (C.D.); European Commission Marie Skłodowska-Curie Fellowship 897672 (H.B.); and NIH NHGRI project HG012544-01 (J.G. and C.D.).

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Authors and Affiliations

Authors

Contributions

H.B. and C.D. conceived of and designed the study. I.C.N., J.R. and T.J.R.K. performed nanopore experiments. J.R. established and troubleshooted the method for POC construction. I.C.N. performed computational analyses of the experimental data. H.B. performed the simulations. C.D. and J.H.G. supervised the work. I.C.N. wrote the initial manuscript draft, and all authors contributed to the writing of the final manuscript.

Corresponding author

Correspondence to Cees Dekker.

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Competing interests

H.B. and C.D. have filed a provisional patent for the nanopore peptide measurement method (NL patent N2024579 P1600131NL00). The remaining authors declare no competing interests.

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Nature Biotechnology thanks Meni Wanunu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Figs. 1–16 and Text 1 and 2.

Reporting Summary

Supplementary Video 1

Video of mean phosphate position within pore for betacatenin variants across many hel308 steps. Derived from MCMC simulation and described in Supplementary Text.

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Nova, I.C., Ritmejeris, J., Brinkerhoff, H. et al. Detection of phosphorylation post-translational modifications along single peptides with nanopores. Nat Biotechnol 42, 710–714 (2024). https://doi.org/10.1038/s41587-023-01839-z

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