Integrated Mass Spectrometry‐based Pipeline for Characterization of Tau in Human Alzheimer’s Brain: Development and Comparison of Sample Preparation Methods

Background Tau protein aggregation into neurofibrillary tangles in the brain is a pathological hallmark of Alzheimer’s disease (AD) and related tauopathies [1]. Though mechanism tau destabilization not fully understood, exhibits several allele-specific isoforms post-translational modifications (PTMs) specific to aggregates [2]. To explore in-depth species tissues AD patients, we developed novel pipelines based on different fractions retrieved by buffer solubility, Filter Aided Sample Preparation (FASP) or immunoprecipitation, before high-resolution mass spectrometry (HRMS) analysis. Method As proof concept, used frozen samples from two patients (0.6-1.2 g per replicate, middle frontal gyrus). The were divided homogenized, ultracentrifuged at 180.000xg for 30min solubilized buffers successively (2 replicates): (1) Sark low-salt buffer, 10% Triton X-100, sarkosyl urea buffer; (2) RIPA high-salt (twice), and, (3) GnH guanidine hydrochloride buffer. Then, performed an antibody-free approach FASP immunoprecipitation with panel commercial antibodies analyzed reversed-phase capillary liquid chromatography, coupled qExactive HRMS. Result HRMS demonstrated that method better than other methods analysis: coverage 63% (full-length, 2N4R) 342 peptide-spectrum matches (PSMs) (Sark: 49% 183 PSMs; GnH: 41% 169 PSMs). PTM-modified (phosphorylated, methylated, acetylated) primarily seen using (59 Sark: 19 PSMs, 21 Immunoprecipitation improved PTM detection compared FASP. Conclusion These findings highlight best performance followed study PTMs Better characterization molecular profile opens gate understanding pathogenesis represents opportunity biomarker field. [1] Mroczko, B. et al. Int J Mol Sci 2019, 20, E4661, https://doi.org/10.3390/ijms20194661. [2] Boyarko, B.; Hook, V. Front. Neurosci. 2021, 15, 702788, https://doi.org/10.3389/fnins.2021.702788.