10.1021/acs.analchem.5b04509.s002 Waltraud Mair Waltraud Mair Jan Muntel Jan Muntel Katharina Tepper Katharina Tepper Shaojun Tang Shaojun Tang Jacek Biernat Jacek Biernat William W. Seeley William W. Seeley Kenneth S. Kosik Kenneth S. Kosik Eckhard Mandelkow Eckhard Mandelkow Hanno Steen Hanno Steen Judith A. Steen Judith A. Steen FLEXITau: Quantifying Post-translational Modifications of Tau Protein <i>in Vitro</i> and in Human Disease American Chemical Society 2016 novel therapeutics site occupancies protein tau assay measure phosphorylation stoichiometry Tau Protein tau PTM landscape tau pathology Human Disease Tauopathies AD pathology FLEXITau 20 phosphorylations 2016-02-15 00:00:00 Dataset https://acs.figshare.com/articles/dataset/FLEXITau_Quantifying_Post_translational_Modifications_of_Tau_Protein_i_in_Vitro_i_and_in_Human_Disease/3085372 Tauopathies, including Alzheimer’s disease (AD), are associated with the aggregation of modified microtubule associated protein tau. This pathological state of tau is often referred to as “hyperphosphorylated”. Due to limitations in technology, an accurate quantitative description of this state is lacking. Here, a mass spectrometry-based assay, FLEXITau, is presented to measure phosphorylation stoichiometry and provide an unbiased quantitative view of the tau post-translational modification (PTM) landscape. The power of this assay is demonstrated by measuring the state of hyperphosphorylation from tau in a cellular model for AD pathology, mapping, and calculating site occupancies for over 20 phosphorylations. We further employ FLEXITau to define the tau PTM landscape present in AD post-mortem brain. As shown in this study, the application of this assay provides mechanistic understanding of tau pathology that could lead to novel therapeutics, and we envision its further use in prognostic and diagnostic approaches for tauopathies.