Single-molecule resolution of post-translational modification on GRK4 with cyclodextrin/SiNx hybrid nanopore.

Abstract

Post-translational modifications (PTMs) on G protein-coupled receptor kinase 4 (GRK4) are implicated in hypertension pathogenesis, their label-free analysis at the single-chemical-group level remains challenging. Here, we construct a γ-cyclodextrin/silicon nitride (γ-CD/SiNx) hybrid nanopore by controlled dielectric breakdown followed by electrophoretic insertion of a single γ-CD molecule into a size-matched SiNx pore, and use it to discriminate PTMs on GRK4-derived oligopeptides. By recording ionic current blockades (ΔI/Io) and dwell times in 1 M KCl at 200 mV, the angstrom-scale hydrophobic cavity of γ-CD provides high spatial resolution: the platform resolves peptides differing by few-residue substitutions and distinguishes palmitoylation, single/multiple phosphorylation, and acetylation on identical 15-residue sequences. Palmitoylated peptides show enhanced capture rates and longer dwell times (2.2 ± 0.1 ms) than unmodified controls (1.0 ± 0.1 ms); mono-phosphorylation reduces normalized current blockade amplitude (ΔI/Io = 0.51 ± 0.02) whereas tri-phosphorylation increases it (ΔI/Io = 0.65 ± 0.02); positional phosphorylation isomers and peptide mixtures are resolved by combined amplitude and dwell-time readout. This label-free single-molecule platform offers a proof-of-concept route toward PTM profiling; validation under physiological conditions and in biological matrices is required before diagnostic application.