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

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.