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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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.).
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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Nova, I.C., Ritmejeris, J., Brinkerhoff, H. et al. Detection of phosphorylation post-translational modifications along single peptides with nanopores. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023-01839-z
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