Large-scale single-molecule analysis of tau proteoforms – Figure 5 with James Joly

In this video, James Joly presents Figure 5 from our recent Nature Methods paper titled, “Large-scale single-molecule analysis of tau proteoforms.” This figure covers the application of the Tau Proteoforms Assay to human samples.

Read the full paper here.

Three takeaways from the video

Today’s phospho-tau biomarkers are measured in ways that miss most of the information. Tau helps build the structural highways that connect neurons, and is dynamically regulated in healthy brains; when regulation breaks down, disease follows. Three phosphorylation sites, pT181, pT217, and pT231, have all been implicated as Alzheimer’s biomarkers, and two of them, pT181 and pT217, are used in FDA-approved diagnostics. But existing technologies measure bulk tau, averaging signals from individual molecules across the whole population. This makes it impossible to tell which molecules a signal came from. They also measure individual phosphorylation events rather than combinations, which means the field has had no way of knowing whether these important markers occur in isolation or together. Iterative Mapping addresses both limitations by interrogating individual tau molecules and mapping their isoform and phosphorylation statuses independently. It effectively shows which molecules carry two, three, four, five, or even up to seven phosphorylation events.

Profiling patient brains revealed distinct, disease-associated tau proteoform signatures. The team profiled tau proteoforms in seven patient brains, five with dementia and two without, and found patient-specific proteoform profiles that correlated with disease status. In the patient with the most severe disease pathology, Iterative Mapping quantified quadruply phosphorylated tau proteoforms, with pT181, pT217, pT231, and pS396 all present on the same tau molecules, at distinctly elevated levels. This is exactly the kind of co-occurrence that bulk measurement cannot establish.

The data suggests tau phosphorylation follows a preferred order. Looking at how likely the various phosphorylation events could be found in combination with one another, a pattern emerged: pT231 and pS396 appear to come first, followed by pT217, with pT181 added last. This apparent cascade is visible only through Iterative Mapping, existing technologies are blind to it. If it holds up, it may eventually help physicians track disease progression, identify which patients are likely to progress to severe disease, and guide both care decisions and the development of new treatments.

Closing

Figure 5 is where the validation work in the earlier figures pays off, moving from controls and models into human disease biology. What James finds most exciting is the potential for this new kind of analysis: proteoforms have never been measurable quantitatively at scale, and putting that capability into other researchers’ hands is what makes the next round of discoveries possible.

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If you’d like to use the Nautilus Tau Proteoforms Assay or our new AKT1 and EGFR Proteoforms Assays in your research, please reach out through the form found on the Iterative Mapping Early Access Program webpage.