Phosphorylated tau is the main protein present in neurofibrillary tangles, the presence of which is a key neuropathological hallmark of Alzheimer’s disease (AD). The toxic effects of phosphorylated tau are likely mediated by interacting proteins; however, methods to identify these interacting proteins comprehensively in human brain tissue are limited. Here, we describe a method that enables the efficient identification of hundreds of proteins that interact with phosphorylated tau (pTau), using affinity purification–mass spectrometry (AP-MS) on human, fresh-frozen brain tissue from donors with AD. Tissue is homogenized using a gentle technique that preserves protein–protein interactions, and co-immunoprecipitation of pTau and its interacting proteins is performed using the PHF1 antibody. The resulting protein interactors are then identified using label-free quantitative liquid chromatography-mass spectrometry (LC-MS)/MS. The Significance Analysis of INTeractome (SAINT) algorithm is used to determine which proteins significantly interact with pTau. This approach enables the detection of an abundance of all 6 isoforms of tau, 23 phosphorylated residues on tau, and 125 significant pTau protein interactors, in human AD brain tissue.
CITATION STYLE
Pires, G., Ueberheide, B., Wisniewski, T., & Drummond, E. (2023). Use of Affinity Purification–Mass Spectrometry to Identify Phosphorylated Tau Interactors in Alzheimer’s Disease. In Methods in Molecular Biology (Vol. 2561, pp. 263–277). Humana Press Inc. https://doi.org/10.1007/978-1-0716-2655-9_14
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