Site-specific post-translational modification detection by polar charged engineered MspA nanopores

Deciphering the complex chemical code of post-translational modifications (PTMs) is fundamental to proteomics but remains challenging due to the low abundance of modified proteins and the difficulty in resolving isobaric positional isomers. Conventional mass spectrometry and affinity-based methods often lack the sensitivity or specificity required to capture this full chemical diversity. Here, we present a general strategy for site-specific PTM recognition based on MspA nanopores engineered with polar charged residues. By systematically tuning the constriction site (position N91) with polar charged residues, we introduced specific electrostatic, electroosmotic flow and steric interactions that dramatically enhance molecular recognition capabilities. This engineered interface enabled the label-free discrimination of 10 distinct PTM types across 26 peptides, including phosphorylation, glycosylation, and-reported here for the first time via nanopore sensing-lysine crotonylation and succinylation. Furthermore, the nanopore is capable of distinguishing subtle positional isomers. Coupled with a machine learning algorithm that classifies single-molecule events with >98% accuracy, our findings establish a generalizable principle of electrostatic gating for PTM profiling, offering a versatile chemical tool for next-generation single-molecule proteomics.