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Large-scale single-molecule analysis of tau proteoforms
Joly J, Budamagunta V, Zhang Z, Nortman B, Jouzi M, Bhatnagar R, Egertson J, Flaster M et al.. Nature methods, 2025-06-27
Topic: Disease mechanisms in lab and animal studies
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.
Key findings
- 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.
- 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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Study details
- Effect size0.1%; 5.5%
- Evidence strengthLow
How this study scored 43/100
Published in a peer-reviewed journal. Scored zero for study design, sample size, studied in people and randomised / blinded because the record does not state it. We have no data on journal's citation record, times cited, citations per year, impact for its field and year, cited by studies here, still being cited, senior author's track record, institution's track record and multi-centre collaboration, so those were left out: it earned 25 of the 58 points we could judge it on, which is 43 out of 100.
| Component | Points | Why |
|---|---|---|
| Journal tier | 7 / 10 | Tier 3 — peer-reviewed journal (Nature methods) |
| Journal's citation record | — / 4 | No journal record from OpenAlex — left out of this score |
| Study design | 0 / 12 | No study design recorded — scores zero, not guessed (not stated in the record — scored 0) |
| Strength of evidence | 1 / 5 | Rated “low” in our summary |
| Peer-review validation | 10 / 10 | Peer-reviewed. Published as an article in a named peer-reviewed journal. |
| How recent | 4 / 5 | 2025 — within 3 year(s) of the newest study here |
| Sample size | 0 / 4 | Participant count not stated — scores zero (not stated in the record — scored 0) |
| Effect reported | 2 / 3 | Effect reported as a number, without an interval or test |
| Studied in people | 0 / 3 | Not stated whether this was studied in people — scores zero (not stated in the record — scored 0) |
| Randomised / blinded | 0 / 2 | No randomisation or blinding stated — scores zero (not stated in the record — scored 0) |
| Times cited | — / 7 | No citation count (not found in OpenAlex) — left out of this score |
| Citations per year | — / 5 | No citation count or no year — left out of this score |
| Impact for its field and year | — / 6 | No field-weighted citation impact (OpenAlex has none yet) — left out of this score |
| Cited by studies here | — / 6 | Not yet checked against this library's citation graph — left out of this score |
| Still being cited | — / 4 | No yearly citation counts — left out of this score |
| Senior author's track record | — / 4 | Senior author's record not available — left out of this score |
| Institution's track record | — / 3 | No institution listed in OpenAlex — left out of this score |
| Multi-centre collaboration | — / 3 | Institutions not listed — left out of this score |
| Indexed in PubMed | 1 / 2 | Not in PubMed, but has a DOI |
| Free to read | 0 / 2 | Behind a paywall — scores zero |
| Points | 25 / 58 | Scored on 11 of 20 parts. We have no data for the rest, so they are left out: 25 of the 58 points we could judge it on is 43 out of 100. |
| Total | 43 / 100 | — |
What each of these 20 components measures, and what this score is not →
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Built from the record alzhub holds for this paper, not looked up. Volume, issue and page numbers are not held in the record. The record's author list ends in “et al.”, so not every author is named. Check the citation against the original before you use it.
APA 7th
Joly, J., Budamagunta, V., Zhang, Z., Nortman, B., Jouzi, M., Bhatnagar, R., et al. (2025). Large-scale single-molecule analysis of tau proteoforms. Nature methods. https://doi.org/10.1101/2025.06.26.660445
Vancouver
Joly J, Budamagunta V, Zhang Z, Nortman B, Jouzi M, Bhatnagar R, et al. Large-scale single-molecule analysis of tau proteoforms. Nature methods. 2025. doi:10.1101/2025.06.26.660445.
BibTeX
@article{joly2025-184e1a12,
author = {Joly, J. and Budamagunta, V. and Zhang, Z. and Nortman, B. and Jouzi, M. and Bhatnagar, R. and Egertson, J. and Flaster, M. and others},
title = {{Large-scale single-molecule analysis of tau proteoforms}},
journal = {Nature methods},
year = {2025},
doi = {10.1101/2025.06.26.660445},
url = {https://doi.org/10.1101/2025.06.26.660445}
}