Large-scale single-molecule analysis of tau proteoforms – Figure 2 with Brittany Nortman

In this video, Brittany Nortman presents Figure 2 from our recent Nature Methods paper titled, “Large-scale single-molecule analysis of tau proteoforms.” This figure covers the adaptation of Iterative Mapping to measure proteoforms of tau.

Read the full paper here.

Three takeaways from the video

Tau hyperphosphorylation is central to Alzheimer’s, but the details are out of reach for most current technologies. When multiple phosphate groups are added across tau, the protein behaves incorrectly, detaching from microtubules and eventually forming the neurofibrillary tangles that are a defining pathology of the disease. Phosphorylation sites are already important diagnostic biomarkers, yet key questions are unanswered: which specific phosphorylations occur together on individual tau molecules at different disease stages, how those molecules are created, and which patterns matter most to disease. Many disease-relevant proteins are likely similar to tau in this respect, generating distinct proteoforms that we have had very few ways to measure.

The Tau Proteoforms Assay resolves those combinations on every protein molecule simultaneously. The assay uses 12 tau antibodies — pan-tau, isoform-specific, and phosphosite-specific probes — giving it the potential to quantify up to 768 proteoform groups. Fluorescent imaging locates every tau molecule on a Voyager Flow Cell, Iterative Mapping repeatedly probes each individual molecule and records whether each antibody binds or doesn’t, and machine learning-based algorithms map the resulting binary binding patterns to specific proteoform identities.

Stepwise validation shows the data can be trusted. The team characterized binding behavior for each antibody using peptides, then moved to recombinant controls: tau 0N3R, tau 2N4R modified with PKA (a kinase that specifically phosphorylates tau at S214), and tau 0N4R modified with ERK2 (a more promiscuous kinase that generates a broader range of proteoforms). Running these controls individually and in mixtures confirmed that the platform accurately detects and quantifies the expected combinations of isoforms and phosphorylation sites. As Brittany puts it, it’s one thing to have a novel platform and another to fully trust the data coming out of it — getting the expected results in a well-controlled system is what makes it possible to move into complex biological systems with confidence.

Up next

In the next video in this series, Maryam Jouzi takes on Figure 3, which covers the in-depth quantitative characterization of the Nautilus Tau Proteoforms Assay. She shares data demonstrating the quantitative power of the assay in depth.

If you want to access the full video series, please fill out the form here:

Access the full series

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.