Application of Open-Search Proteomics for Comprehensive and Accurate Profiling of Known Tau PTMs and Characteristic Mass Shifts in Alzheimer’s Disease

Tau is the primary protein studied in Alzheimer's disease (AD), and its post-translational modifications (PTMs) play a critical role in disease progression. However, conventional closed-search PTM workflows are limited to predefined modification types, which constrains the detection of unexpected Tau PTMs in AD. In this study, we generated a high-resolution delta-mass table by combining known PTMs from Open-pFind with a dense series of additional mass delta values and applied an open-search proteomics workflow to analyze Alzheimer's disease data. This approach identified 23 Tau PTM sites in AD, primarily involving ubiquitination (Ub) and deamidation (Asn → Asp). These characteristic PTM sites enabled more accurate discrimination of Braak stage V-VI samples not only from controls but also from earlier Braak stages. We further established a unified AD-identifying indicator based on ubiquitination, deamidation, and characteristic mass shifts, which accurately classified AD samples in data set PXD020517 & PXD020538 and was validated in PXD038901. In addition, we detected some previously unreported Tau PTMs and characteristic mass shifts through open-search analysis. In conclusion, we have successfully employed Open-pFind along with a high-resolution delta-mass table to achieve a comprehensive analysis of both known Tau PTMs and characteristic mass shifts to accurately distinguish Alzheimer's disease samples.