Mapping phosphorylation post-translational modifications along single peptides with nanopores

Current methods to detect post-translational modifications (PTMs) of proteins, such as phosphate groups, cannot measure single molecules and often cannot differentiate between closely spaced phosphorylation sites. Using a nanopore sequencing approach, we here report detection of PTMs at the single-molecule level on immunopeptide sequences with cancer-associated phosphate variants. We reliably discriminate peptide sequences with one or two closely spaced phosphates with 95% accuracy for individual reads of single molecules.