PTM-Driven Reshaping of the Peptide Translocation Landscape in Bilayer Graphene Nanopores

Post-translational modifications (PTMs) underpin much of protein regulation, yet their single-molecule readout remains a challenge in nanopore proteomics. While biological nanopores have shown exquisite PTM sensitivity, the microscopic mechanisms by which PTMs perturb signals in solid-state nanopores are largely unexplored. Here, we use all-atom molecular dynamics to investigate how three common PTMs, acetylation, phosphorylation, and methylation, modulate the translocation of a cancer-relevant p53 peptide fragment through a bilayer graphene nanopore. We find that PTMs remodel the translocation landscape far more strongly at the level of dwell-time statistics than at the level of mean current blockade. Acetylation enhances peptide-graphene adhesion and substantially slows transport, with adjacent acetylations producing the longest residence times due to cooperative interfacial interactions, while remotely spaced acetylations yield broader, heterogeneous dynamics. Phosphorylation introduces a negative charge that increases dwell time through an electrostatic tug-of-war, while also generating the largest current blockade among the PTMs studied. In contrast, methylation minimally perturbs translocation due to weak pore interactions and preserved charge. Combining dwell time with relative blockade features enables a simple linear SVM classifier to reliably distinguish unmodified, acetylated, and phosphorylated states. These results establish mechanistic design principles for PTM detection using solid-state nanopores and delineate which classes of PTMs are the most amenable to single-molecule detection with these devices.