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

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