PALM-Scan enables single-molecule site-specific detection of protein S-palmitoylation with a nanopore.

Protein S-palmitoylation is a dynamic and reversible post-translational modification (PTM) that governs diverse cellular processes, yet its study remains constrained by the indirect and ensemble-averaging nature of classical biochemical assays. Here, we report a nanopore-based platform, PALM-Scan (palmitoylation analysis via label-free monitoring and signature capture on nanopore) that directly transduces physicochemical signatures of S-palmitoylated peptides into digital electrical signals. Using an engineered Mycobacterium smegmatis porin A (M2-MspA) nanopore, PALM-Scan enables sequence-independent identification of S-palmitoylation and simultaneously differentiates S-palmitoylation from other cysteine-directed PTMs, including S-nitrosylation and S-glutathionylation, without probes or enrichment. We further demonstrate its translational potential by successfully detecting and distinguishing S-palmitoylated subpopulations of disease-relevant biomarkers, including mouse beclin-1 (BECN1)- and glial fibrillary acidic protein (GFAP)-derived peptides, in complex mixtures with minimal sample input. In summary, by integrating label-free operation, single-molecule resolution, and multiplex discrimination, PALM-Scan provides a transformative tool for interrogating S-palmitoylation dynamics in both basic research and biomedical applications.