Location of Phosphorylation Sites within Long Polypeptide Chains by Binder-Assisted Nanopore Detection

The detection and mapping of protein phosphorylation sites are essential for understanding the mechanisms of various cellular processes and for identifying targets for drug development. The study of biopolymers at the single-molecule level has been revolutionized by nanopore technology. In this study, we detect protein phosphorylation within long polypeptides (>700 amino acids), after the attachment of binders that interact with phosphate monoesters; electro-osmosis is used to drive the tagged chains through engineered protein nanopores. By monitoring the ionic current carried by a nanopore, phosphorylation sites are located within individual polypeptide chains, providing a valuable step toward nanopore proteomics.