Sub-dalton Discrimination of Citrullination by a Semi-rationally Designed Aerolysin Nanopore

Citrullination, a post-translational modification (PTM) with a sub-dalton mass shift of only + 0.984 Da, plays critical roles in epigenetics and autoimmunity and is a hallmark of several inflammatory diseases, but remains a particularly long-standing challenge to resolve by conventional proteomics. Here we present Physical Interaction Transduction on Nanopores (PIToN), a semi-rational model for relating local pore–analyte interactions to nanopore discrimination. Guided by PIToN, we engineered an aerolysin nanopore carrying the K238E mutation, which together with E258 forms a localized electrostatic constriction that functions as a precision “reading head.” This pore enabled label-free, single-molecule detection of peptide citrullination with 98.3% accuracy and distinguished citrullination from methylation-related histone peptide states. The platform also enabled real-time monitoring of enzymatic citrullination and voltage-dependent selective peptide enrichment with up to 99.7% purity. This word provides a practical route for physico-chemically guided nanopore design and broader applications in PTM analysis and protein characterization.