Single-molecule profiling of full-length Tau isoforms and phosphorylated proteoforms

Abnormal post-translational modifications (PTMs) can lead to the aggregation of Tau proteins, which characterize a wide range of neurodegenerative diseases known as tauopathies. Some abnormal Tau proteins will be released into biological fluids, providing a chance for minimally- or non-invasive diagnosis. For Alzheimer's Disease (AD), site-specific phosphorylations and truncations are representative PTMs that have been proved as biomarkers for its early diagnosis and progression monitoring. To detect and quantify these abnormal Tau proteoforms, two methodologies—immunoassays and mass spectrometry (MS)—are frequently employed. However, both of them encounter significant challenges in clinical trials; immunoassays suffer from limited epitopes and false positive results, while MS involves a complicated and time-consuming workflow.Nanopore technology, distinguished by its capability of real-time single-molecule sensing, has shown great prospects in polypeptide analysis, potentially offering distinct advantages over the forementioned methods in clinical trials. Recent progress has been made to sense PTMs deep within full-length polypeptides. By leveraging the recent advances in nanopore technology and the development of numerous site-specific anti-Tau binders, this project aims to exploit a single-molecule toolkit for detecting Tau of varying lengths and clinically relevant phosphorylations across full-length Tau proteoforms. To achieve that, we will leverage engineered nanopores developed by the host lab to facilitate translocation of intact Tau proteoforms, alongside a binder-assisted strategy to amplify the signals of targeted truncations and phosphorylations while minimizing interference from non-target backgrounds. Should our goal accomplished, it could provide a sensitive and robust diagnostic tool for the assessment and monitoring of tauopathies, as well as contribute to the advancement of tauopathies research to single-molecular resolution.