Large-scale single-molecule analysis of tau proteoforms
4 affiliations
- 1Nautilus Biotechnology, San Carlos, CA, USA
- 2Genentech, South San Francisco, CA, USA
- 3Neural Stem Cell Institute, Albany, NY, USA
- 4The Ronald Loeb Center for Alzheimer's Disease at Mt. Sinai, New York, NY, USA
Abstract
Proteins exist as diverse proteoforms resulting from a combination of genetic variation, alternative splicing, and post-translational modifications. Current methods struggle to capture this complexity at the single-molecule level. Here we introduce Iterative Ma pping of P roteoforms (IMaP), a method that enables the massively-parallel interrogation of millions to billions of single-protein molecules through iterative probing with fluorescently labeled antibodies. Using 12 site-specific antibodies, the method is capable of measuring 2 12 (4,096) potential proteoform groups. We used IMaP to measure proteoform group profiles of the tau protein, a key player in neurodegenerative diseases, using two pan anti-tau antibodies (Tau-13, Tau-216), three isoform-specific antibodies (Anti-0N, Anti-2N, Anti-4R), and seven phosphosite-specific antibodies (Anti-pT181, Anti-pS202+pT205, Anti-pT205, Anti-pS214, Anti-pT217, Anti-pT231, and Anti-pS396). The method demonstrates high sensitivity (detecting proteoforms at 0.1% abundance), high reproducibility (median CV <5.5%), and broad dynamic range (>3 orders of magnitude), outperforming conventional techniques in resolving closely related proteoform groups. We demonstrated that the method can be used on relevant biological samples by examining various neuronal models (iNeuron cells, organoids, MiBrains, and mouse brains) and human samples. This examination revealed 130 distinct tau proteoform groups with as many as six phosphorylation events. The non-random distribution of these phosphorylation events suggests ordered and site-specific modification processes rather than random, stochastic accumulation. Certain combinations of phosphorylation events were more abundant than others; for example, pT217 preferentially co-occurred with pT181. In validating the applicability of the assay to human disease samples, we noted a specific pattern of multiple phosphorylation events in an advanced Alzheimer’s disease patient that suggests a sequential pathway of pathological tau modification. Iterative Mapping of Proteoforms provides insights into proteoform complexity at the single-molecule level, with significant implications for understanding protein regulation in neurodegenerative diseases and beyond.
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BibTeX
@article{joly2026largescale,
title = {{Large-scale single-molecule analysis of tau proteoforms}},
author = {James H. Joly and Vivekananda Budamagunta and Zhengjian Zhang and Brittany Nortman and Maryam Jouzi and Rajat Bhatnagar and Jarrett D. Egertson and Michael E. Flaster and Rob Grothe and Sanjib K Guha and Kota Kaneshige and Kevin McVey and Nicholas Nelson and Rukshan T. Perera and Steven J. Tan and Tuan Trinh and David Arnott and Joanna Lipka and Nikhil Janak Pandya and Lionel Rougé and Timothy J. Wendorff and Donald S. Kirkpatrick and Alexis L. Rohou and David C. Butler and Steven Lotz and A Forton and Emily Ruth Sartori and Jessica E. Schwarz and Phillip S. Brereton and Kevin Chen and Michael A. Darcy and HamidReza Golnabi and Ross Hartley and PF Indermuhl and Christina E. Inman and Kevin M. Jin and Stanislav Katsyuk and Rajesh Kota and Bryant Lowry and Jonathan C. Menger and Denise A. Miller and Maureen R. Newman and Adedeji Daniel Ogunyemi and Julia K. Robinson and Noah Steiner and Juanfeng Sun and Scott M. Tabakman and Lisen Wang and Zhiqiang Wang and Sheri K. Wilcox and Nautilus Biotechnology and Jamie E. Anderson and Fletcher E. Bain and Filip Bartnicki and Issa Beekun and Giovanni Bellesia and Rajat Bhatnagar and Keith Bjornson and Phillip S. Brereton and Stephen Brown},
journal = {Nature Methods},
year = {2026},
month = sep,
doi = {10.1038/s41592-026-03188-6},
url = {https://doi.org/10.1038/s41592-026-03188-6},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Large-scale single-molecule analysis of tau proteoforms
AU - James H. Joly
AU - Vivekananda Budamagunta
AU - Zhengjian Zhang
AU - Brittany Nortman
AU - Maryam Jouzi
AU - Rajat Bhatnagar
AU - Jarrett D. Egertson
AU - Michael E. Flaster
AU - Rob Grothe
AU - Sanjib K Guha
AU - Kota Kaneshige
AU - Kevin McVey
AU - Nicholas Nelson
AU - Rukshan T. Perera
AU - Steven J. Tan
AU - Tuan Trinh
AU - David Arnott
AU - Joanna Lipka
AU - Nikhil Janak Pandya
AU - Lionel Rougé
AU - Timothy J. Wendorff
AU - Donald S. Kirkpatrick
AU - Alexis L. Rohou
AU - David C. Butler
AU - Steven Lotz
AU - A Forton
AU - Emily Ruth Sartori
AU - Jessica E. Schwarz
AU - Phillip S. Brereton
AU - Kevin Chen
AU - Michael A. Darcy
AU - HamidReza Golnabi
AU - Ross Hartley
AU - PF Indermuhl
AU - Christina E. Inman
AU - Kevin M. Jin
AU - Stanislav Katsyuk
AU - Rajesh Kota
AU - Bryant Lowry
AU - Jonathan C. Menger
AU - Denise A. Miller
AU - Maureen R. Newman
AU - Adedeji Daniel Ogunyemi
AU - Julia K. Robinson
AU - Noah Steiner
AU - Juanfeng Sun
AU - Scott M. Tabakman
AU - Lisen Wang
AU - Zhiqiang Wang
AU - Sheri K. Wilcox
AU - Nautilus Biotechnology
AU - Jamie E. Anderson
AU - Fletcher E. Bain
AU - Filip Bartnicki
AU - Issa Beekun
AU - Giovanni Bellesia
AU - Rajat Bhatnagar
AU - Keith Bjornson
AU - Phillip S. Brereton
AU - Stephen Brown
PY - 2026
DA - 2026/09/01
JO - Nature Methods
DO - 10.1038/s41592-026-03188-6
UR - https://doi.org/10.1038/s41592-026-03188-6
AB - Proteins exist as diverse proteoforms resulting from a combination of genetic variation, alternative splicing, and post-translational modifications. Current methods struggle to capture this complexity at the single-molecule level. Here we introduce Iterative Ma pping of P roteoforms (IMaP), a method that enables the massively-parallel interrogation of millions to billions of single-protein molecules through iterative probing with fluorescently labeled antibodies. Using 12 site-specific antibodies, the method is capable of measuring 2 12 (4,096) potential proteoform groups. We used IMaP to measure proteoform group profiles of the tau protein, a key player in neurodegenerative diseases, using two pan anti-tau antibodies (Tau-13, Tau-216), three isoform-specific antibodies (Anti-0N, Anti-2N, Anti-4R), and seven phosphosite-specific antibodies (Anti-pT181, Anti-pS202+pT205, Anti-pT205, Anti-pS214, Anti-pT217, Anti-pT231, and Anti-pS396). The method demonstrates high sensitivity (detecting proteoforms at 0.1% abundance), high reproducibility (median CV <5.5%), and broad dynamic range (>3 orders of magnitude), outperforming conventional techniques in resolving closely related proteoform groups. We demonstrated that the method can be used on relevant biological samples by examining various neuronal models (iNeuron cells, organoids, MiBrains, and mouse brains) and human samples. This examination revealed 130 distinct tau proteoform groups with as many as six phosphorylation events. The non-random distribution of these phosphorylation events suggests ordered and site-specific modification processes rather than random, stochastic accumulation. Certain combinations of phosphorylation events were more abundant than others; for example, pT217 preferentially co-occurred with pT181. In validating the applicability of the assay to human disease samples, we noted a specific pattern of multiple phosphorylation events in an advanced Alzheimer’s disease patient that suggests a sequential pathway of pathological tau modification. Iterative Mapping of Proteoforms provides insights into proteoform complexity at the single-molecule level, with significant implications for understanding protein regulation in neurodegenerative diseases and beyond.
SN - 1548-7091
SN - 1548-7105
ER -