Large-scale single-molecule analysis of tau proteoforms – Figure 3 with Maryam Jouzi

In this video, Maryam Jouzi presents Figure 3 from our recent Nature Methods paper titled, “Large-scale single-molecule analysis of tau proteoforms.” This figure covers the accuracy, reproducibility, and dynamic range of the Tau Proteoforms Assay.

Read the full paper here.

Three takeaways from the video

Detecting proteoforms isn’t enough — they have to be quantified. Proteins are never static single entities. They vary in sequence, carry post-translational modifications, exist as different isoforms, and their function in the cell depends on which variations are present and at what abundance. Figure 2 showed that the Nautilus Voyager™ Platform can detect those variations. Figure 3 asks how well it can measure them, focusing on three questions: how accurate the measurements are, how reproducible they are, and across what range of abundances they hold up.

Against known ground truth, the assay is both accurate and highly reproducible. Using tau protein controls as ground truth, the team stress-tested accuracy and measured a mean absolute percent error of less than 10%. Reproducibility was evaluated across variables including different instruments and different runs, yielding a median coefficient of variation of ≤5.5% — tight performance relative to existing methods for proteoform measurement. The assay also quantifies down to 1,000 counts per million, or 0.1% abundance, which sets its limit of quantification. That matters because therapeutic development and mechanistic work on specific modifications both depend on being confident that a measurement made in one lab on one device holds up in another, and many current technologies, including mass spectrometry, have difficulty achieving this kind of quantitative power.

Reproducibility holds all the way down to the lowest measurable abundances. Many existing technologies degrade in measurement quality as abundance drops. The team initially characterized reproducibility only for proteoforms at 5% abundance or higher, and Maryam describes the moment they decided to check whether the reproducibility held at 0.1% as a genuinely nerve-wracking one — they expected it might require substantial additional optimization. Instead, the data showed the same tight CV at 0.1% as at 5% and above. Combined with the platform’s single-molecule resolution, that consistency across a wide dynamic range is what makes the assay suitable for studying low-abundance proteoforms.

Up next

In the next video in this series, Vivek Budamagunta turns from assay characterization to application, examining the landscape of tau proteoforms in models of neurodegenerative disease. Having established that the measurements are accurate, reproducible, and quantitative across a broad range of abundances, the work ahead puts Iterative Mapping to use in studying biology itself.

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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.