Mapping the Landscape of Tau Post-translational Modifications and Kinase-Specific Phosphorylation Signatures by Orthogonal CZE-MS/MS and NanoRPLC-MS/MS

Abstract Hyperphosphorylation of tau is central to the development and progression of Alzheimer’s disease (AD) and other tauopathies. How different kinases and their phosphorylated tau products, as well as additional post-translational modifications (PTMs) of tau, contribute to AD is a critical yet poorly understood question. We created a comprehensive landscape of PTMs (i.e., phosphorylation, acetylation, and methylation) of tau across isoforms (2N4R, 1N4R, and 0N3R) and kinases (GSK3β, TTBK1, and DYRK1A) using complementary capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) and nanoflow reversed-phase liquid chromatography (nanoRPLC)-MS/MS. Kinase-treated 2N4R (Tau-441) yielded 65 phosphorylation sites in total, consisting of 31 shared sites and additional kinase-specific subsets; 15 phosphosites were observed only in kinase-treated samples. All kinases generated the AT8 (Ser202/Thr205) and PHF-1 (Ser396/Ser404) epitopes. Across all conditions and isoforms, we identified 76 unique phosphorylation sites on Tau-441. Orthogonal separations provided near-complete Tau-441 sequence coverage (CZE: 96.3 ± 2.5%; RPLC: 95.9 ± 2.8%; n = 12 runs each) with phosphorylation sites detected uniquely by either platform. In addition, 49 Lys/Arg residues were acetylated and/or methylated (including K174, K274, and K281). This work revealed convergent and kinase-specific tau PTM patterns relevant to AD mechanisms and kinase prioritization. We expect the comprehensive kinase-specific PTM resource of tau generated here will benefit future tau-focused AD research.