Tyrosine sulfation plays a crucial role in modulating protein-protein interactions and has been implicated in a wide range of physiological and pathological processes. Despite being estimated as the most abundant post-translational modification (PTM) of tyrosine—occurring in approximately 1% of all tyrosine residues—only about 160 tyrosine-sulfated proteins have been experimentally identified over the nearly 70 years of research. Therefore, tyrosine sulfation remains one of the least characterized PTMs, highlighting the need for a direct and efficient method for accurate detection.
Here, we present a nanopore-based sequence-independent strategy to directly map and quantify tyrosine sulfation in native peptides. Molecular dynamics simulations and nanopore mutations reveal specific interactions between sulfated tyrosine and the engineered nanopore, enabling precise determination of both its quantity and location. By using this strategy, we demonstrate the discovery of tyrosine sulfation in peptide mixtures, including unknown peptide fragments derived from native proteins. Furthermore, this approach allows for the sequencing of sulfated peptide fragments by correlating peptide mass with the enhanced current blockade caused by sulfation, as established through our method. Additionally, we show that this technique can detect tyrosine sulfation in peptides with ultra-low abundance—down to 1%—and distinguish it from isobaric phosphorylation at the same site. This sequence-independent strategy for directly identifying tyrosine sulfation demonstrates the potential of nanopore technology to investigate specific PTMs in native proteins from real-world samples, with implications for omics-level research.
The related paper entitled “Direct mapping of tyrosine sulfation states in native peptides by nanopore” has been published on Nature Chemical Biology on September 25, 2024 (Paper link:https://www.nature.com/articles/s41589-024-01734-x, DOI: 10.1038/s41589-024-01734-x). Dr. Meng-Yin Li and Prof. Yi-Tao Long from our department are the co-corresponding authors. PhD. student Hongyan Niu and Dr. Meng-Yin Li are the co-first authors. Thanks for Prof. Chunmao He from South China University of Technology for kindly supplying HIRV1 protein. This research was supported by the National Natural Science Foundation of China and the Program for Outstanding PhD Candidates of Nanjing University.
Figure 1. Single-channel recording for mapping the sulfation states of tyrosine with mutant aerolysin nanopore.
Figure 2. The molecular mechanism of T232K mutant aerolysin mapping the tyrosine sulfation states by MD Simulations.
Figure 3. Determining the tyrosine sulfation without the influences of peptide sequences.
Figure 4. Discovering tyrosine sulfation in peptide mixture and native protein.
Figure 5. Discrimination of sulfation and phosphorylation on same tyrosine.