Abstract
Sulfation is considered the most prevalent post-translational modification (PTM) on tyrosine; however, its importance is frequently undervalued due to difficulties in direct and unambiguous determination from phosphorylation. Here we present a sequence-independent strategy to directly map and quantify the tyrosine sulfation states in universal native peptides using an engineered protein nanopore. Molecular dynamics simulations and nanopore mutations reveal specific interactions between tyrosine sulfation and the engineered nanopore, dominating identification across diverse peptide sequences. We show a nanopore framework to discover tyrosine sulfation in unknown peptide fragments digested from a native protein and determine the sequence of the sulfated fragment based on current blockade enhancement induced by sulfation. Moreover, our method allows direct observation of peptide sulfation in ultra-low abundance, down to 1%, and distinguishes it from isobaric phosphorylation. This sequence-independent strategy suggests the potential of nanopore to explore specific PTMs in real-life samples and at the omics level.
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Data availability
Data supporting the findings of this study are available in the main text and the Supplementary Information. Additional raw data and the aerolysin cryo-EM structure are available at figshare (https://doi.org/10.6084/m9.figshare.26494222). Source data are provided with this paper.
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Acknowledgements
We thank C. He from South China University of Technology for supplying HIRV1 protein, X.Y. Wu for help in aerolysin construction and H. Bhatti for discussions in writing. This research was supported by the National Natural Science Foundation of China (22334006 to Y.-T.L., 22027806 to Y.-T.L. and 22207054 to M.-Y.L.), the programs for high-level entrepreneurial and innovative talents introduction of Jiangsu Province, and the Program for Outstanding PhD Candidates of Nanjing University (202401A05 to H.N.).
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M.-Y.L., H.N. and Y.-T.L. conceived the idea and designed the experiments. H.N. performed the nanopore experiments. H.N. and Y.G. analyzed the nanopore data. H.N. constructed the MD simulations, with assistance from M.-Y.L. H.N., J.-G.L., J.J. and Y.-L.Y. expressed and purified protein nanopores. H.N. and M.-Y.L. interpreted the data and wrote the manuscript. M.-Y.L. and Y.-T.L. supervised the project. All authors critically reviewed the manuscript.
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Sequences of primers for aerolysin mutations
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Niu, H., Li, MY., Gao, Y. et al. Direct mapping of tyrosine sulfation states in native peptides by nanopore. Nat Chem Biol 21, 716–726 (2025). https://doi.org/10.1038/s41589-024-01734-x
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DOI: https://doi.org/10.1038/s41589-024-01734-x
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