Large-scale single-molecule analysis of tau proteoforms – Figure 1 with Zhengjian Zhang

In this video, Zhengjian Zhang presents Figure 1 from our recent Nature Methods paper titled, “Large-scale single-molecule analysis of tau proteoforms.” This figure covers the single-molecule Iterative Mapping of Proteoforms workflow.

Read the full paper here.

Three takeaways from the video

Iterative Mapping turns a protein sample into an ordered array of individually addressable molecules. Each protein is covalently attached to a proprietary nanoparticle at one-to-one stoichiometry, and because the nanoparticles are designed to match the size and geometry of the landing pads on the Voyager flow cell, the conjugates self-assemble into arrays of billions of distinguishable single molecules. Those molecules can be probed at single-molecule resolution, imaged, and independently tracked across hundreds of analysis cycles in parallel.

Proteoform identity is built up cycle by cycle. An array of proteins from a single gene is repeatedly probed with fluorescently labeled reagents that bind isoform-defining features and site-specific post-translational modifications. The unique combination of features detected on each molecule defines its proteoform, and molecules sharing an identity are tallied to quantify the proteoform landscape. Unlike conventional mass spectrometry, which cannot localize many features back to individual protein molecules, the Voyager™ Platform interrogates full proteins as single units and localizes every probe binding event to a specific molecule.

The Tau Proteoforms Assay shows how sensitive the approach can be. Tau is enriched from cell and tissue extracts with bead-immobilized monoclonal antibodies, derivatized with mTz groups for click chemistry, conjugated to an excess of nanoparticles while still bound to the enrichment beads, and then gently eluted using competitive peptides that mimic tau epitopes. Zhengjian shares an early result from development in which one microgram of mouse brain extract was diluted 100-fold into tau-free extract and still yielded enough nanoparticles for on-platform detection — a hint at the low-abundance, low-input applications this opens up.

Up next

In the next video in this series, Brittany Nortman shares initial data from the paper showing that Iterative Mapping can effectively quantify tau proteoforms — work that may substantially expand our understanding of the mechanisms underlying Alzheimer’s disease and other tau pathologies. As Zhengjian notes, this is just the beginning: the general method is designed to be readily adaptable to proteoforms of any protein of interest, as well as to broadscale proteomic analysis.

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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.