Abstract
Tau protein aggregation is associated with posttranslational modifications (PTMs) in 75% of all dementia cases. The distribution of tau pathology and the presence of specific tau phosphorylation sites of interest are typically visualized and measured using antibodies. However, previous knowledge of the target epitopes is required. Additionally, antibodies can be used in a semi-quantitative manner but cannot be used to determine the absolute amount of tau or the extent of the modifications at specific sites or domains. Here we present a discovery assay that characterizes the global qualitative and quantitative tau modification landscape of a sample without a priori knowledge. Our workflow uses sarkosyl fractionation to extract the pathological tau species from sample-limited brain specimens, followed by mass spectrometry (MS) to characterize and quantify tau PTMs. The two-step MS-based proteomics approach includes an exploratory tau PTM analysis and a targeted full-length expressed stable isotope-labeled tau assay, which monitors specific unmodified tau peptides using a heavy isotope-labeled internal standard as a reference. This enables the absolute quantification of the respective tau peptides and the total tau amount in the sample, thus providing the modification extent of tau PTMs. This approach provides precise, comprehensive, qualitative and quantitative tau PTM profiling of the sample. It also enables the detailed molecular comparison of tau across multiple experiments, including a comparison between neurodegenerative diseases, stages of the disease, human patient heterogeneity and characterization of animal models. The approach is useful for studying the molecular features of pathological tau in neurodegeneration. The procedure requires 7–8 d and is suitable for users with expertise in targeted and untargeted MS-based protein analysis.
Key points
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A biochemical approach for the characterization of pathological protein aggregates, in which an untargeted mass spectrometry proteomics approach is used to map tau modifications, followed by a full-length expressed stable isotope-labeled tau (FLEXITau) assay that monitors unmodified tau peptides in a targeted manner, enabling posttranslational modification (PTM) quantification.
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The approach improves on the use of targeted antibodies for detecting specific modifications as it can be used without prior knowledge of either PTM type or PTM site.
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References
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Acknowledgements
This work was supported by grants from the National Institutes of Health (R01 AG071858 and U01 NS110438). We thank the Rainwater Charitable Foundation and the Ellison Foundation for their interest and support of tauopathy research.
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Contributions
Methodology: K.W., A.V., M.K., H.S. and J.A.S. Writing: K.W., A.V., M.K., H.S. and J.A.S. Visualization: K.W. and J.A.S. Funding acquisition: J.A.S. All authors read and approved the final manuscript.
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Nature Protocols thanks Cristian Lasagna-Reeves, Martin Larsen and Danielle Swaney for their contribution to the peer review of this work.
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Key references using this protocol
Mair, W. et al. Anal. Chem. 88, 3704–3714 (2016): https://doi.org/10.1021/acs.analchem.5b04509
Wenger, K. et al. Mol. Neurodegener. 18, 10 (2023): https://doi.org/10.1186/s13024-023-00601-y
Wesseling, H. et al. Cell 183, 1699–1713.e13 (2020): https://doi.org/10.1016/j.cell.2020.10.029
Dujardin, S. et al. Nat. Med. 26, 1256–1263 (2020): https://doi.org/10.1038/s41591-020-0938-9
Supplementary information
Supplementary Information
Supplementary Fig. 1.
Supplementary Table 1
Transition list including monitored precursors and fragments of the FLEXITau assay.
Supplementary Data 1
Sequence of FLEXITau expression vector and the corresponding protein product.
Supplementary Data 2
Skyline library for FLEXITau data evaluation containing all monitored precursors and fragments of the FLEXITau assay.
Supplementary Data 3
Example for evaluating FLEXITau data, including the calculation of absolute total tau amount and the relative modification extent.
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Wenger, K., Viode, A., Kumar, M. et al. Quantitative profiling of posttranslational modifications of pathological tau via sarkosyl fractionation and mass spectrometry. Nat Protoc 19, 1235–1251 (2024). https://doi.org/10.1038/s41596-023-00939-z
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DOI: https://doi.org/10.1038/s41596-023-00939-z
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