Simultaneous Profiling and Quantification of Tau and Its Phosphorylated Isomers Using Engineered FraC Nanopores

Tau aggregation and hyperphosphorylation are key pathological hallmarks and early diagnostic biomarkers of Alzheimer's disease (AD). However, the simultaneous profiling and quantitative analysis of Tau and its phosphorylated isoforms remain technically challenging. Here, by introducing an acidic aspartate residue at the G6 site of FraC^G13F, we engineered a high-resolution FraC^G6DG13F nanopore with enhanced temporal resolution for protonated polypeptide detection. Integrated with machine-learning algorithms, our nanopore-based workflow enabled the real-time identification of 12 tryptic peptides from native Tau (2N4R) protein with over 91.1% accuracy. Furthermore, positional phosphorylation isomers at either S400 or S402 of Tau (2N4R) were precisely resolved and quantified in mixed digestion samples, achieving classification accuracy exceeding 93% and sensitivity comparable to that of microgram-level mass spectrometry. This study demonstrates a promising advancement of global analysis of phosphorylated Tau in nanopore-based methods, highlighting its potential for high-resolution characterization and accurate quantification of proteins with PTMs.