NEW YORK – Proteomics firm Nautilus Biotechnology has generated data indicating that its proteomics platform can detect around 800 proteoforms of the Alzheimer’s disease-associated protein tau.
The work, which the company detailed in a presentation at the annual meeting of the Human Proteome Organization (HUPO) in October, indicates that a large swath of proteoform biology remains uncharted and suggests the role that targeted single-molecule approaches could play in analyzing it.
Nautilus' platform uses chips functionalized with DNA origami structures that allow researchers to deposit single proteins in extremely dense arrays, enabling single-molecule analysis of as many as 10 billion individual proteins. Ultimately, the company hopes to use iterative probing of these proteins with semi-specific affinity agents to enable proteome-scale analyses. In the meantime, however, it has focused on more targeted experiments, using antibodies to specific posttranslational modifications to identify modified forms of proteins of interest.
At the US HUPO annual meeting in March, Nautilus shared data from an analysis of tau proteoforms in which company researchers created samples with four different tau proteoforms, mixed in different ratios, and used the platform to identify them and establish their proportion. The platform also detected unexpected tau proteoforms that were created when the reactions used to produce the expected proteoforms failed to proceed to completion.
At the October HUPO meeting, Nautilus presented an expanded assay using a series of 11 antibodies that the company said could, in theory, identify as many as 2,048 tau proteoforms. In fact, the researchers detected around 40 percent, or roughly 800, of these potential proteoforms, according to Parag Mallick, Nautilus's cofounder and chief scientist.
Though the company has not published data from this work, the results suggests there could be a vast landscape of proteoforms that has largely gone unexplored to date.
Neil Kelleher, a professor of chemistry at Northwestern University and a pioneer in top-down proteomics and proteoform research, said the Nautilus platform's potentially high dynamic range could allow it to identify lower abundance proteoforms that have remained out of reach of technologies like mass spectrometry.
Kelleher said his lab is typically able to identify proteoforms down to 1 percent relative abundance using mass spec. He suggested that the Nautilus platform, with its densely populated single-molecule arrays, will likely be able to go even deeper than that.
Mallick declined to specify the dynamic range across which the company detected the roughly 800 tau proteoforms. However, he said that early studies suggest it will be able to detect proteoforms across a broader dynamic range than is possible with mass spec.
The Nautilus researchers analyzed three sample types — organoid models, miBrain models (multicellular brain models based on stem cells), and mouse models of Alzheimer's disease. Mallick said they found roughly the same number of proteoforms across each sample type. While the researchers saw more multiply phosphorylated species of tau in the organoid and miBrain models compared to the mouse brain, this manifested as an increase in proteoform abundance, not diversity, he added.
Mallick noted the preliminary nature of the study and said that more definitively establishing the proteoform landscape of the different sample types would require a larger number of samples, time points, and replicates.
Sally Temple, scientific director of the Neural Stem Cell Institute in Rensselaer, New York, said that the platform's potential for delving deep into proteoform populations could "reveal so much more biology than we have now."
Temple is a member of the Tau Consortium, an organization funded by the Rainwater Charitable Foundation to research tau biology and tauopathies like frontotemporal dementia and Alzheimer's disease. As part of this work, she and her colleagues have developed brain organoid models using cell lines from frontotemporal dementia patients with mutations in the MAPT gene, allowing them to study the impact of those mutations.
"I think what Nautilus recognized is that this is a system for them to test their technology out because we do see a variety of tau pathologies," she said, noting that one consequence of MAPT mutations is an increase in phosphorylated forms of tau.
Temple said she and her colleagues have sent Nautilus several rounds of organoids to analyze. The data they have seen has validated previous findings such as an increase in phosphorylated tau in certain mutant lines. The researchers are also seeing "a great variety of new tau proteoforms" with the technology, she said.
"I think this is just the beginning of really understanding what these different tau molecules are," she said. "The excitement for us is that we are seeing this at single-molecule resolution, and we have the opportunity, even with just the antibody sets [Nautilus] has now, to understand a lot about the impact of mutation. And going down the road, as they add to that collection, that increased complexity will tell us even more about how tau is modified and what the consequence of those modifications could be."
"Phosphorylation is very important for the creation of [tau] aggregates that are a critical pathological process, and if we can understand that with greater granularity … we can identify new ways of combatting this process," Temple said.
Looking ahead, she said she hopes to work with Nautilus to look at how tau proteoform profiles compare across samples featuring different mutations, as well as to look at how tau proteoform populations change over the course of tauopathies.
The ability to detect hundreds to thousands of proteoforms of a single protein raises the question of how researchers might begin to make sense of this information.
Particularly with very low abundance proteoforms, their meaning is unknown, Kelleher said. "Is it biology, is it a lack of control, is it part of the disease process? I think the jury is still out."
Technologies like the Nautilus platform, as well as advances in other areas like mass spec, could produce tools so that "at least we can ask those questions," Kelleher said.
"Some of it may be noise," Temple said. "But, guided by what has been done to date, I think this window into posttranslational modifications is going to have huge value for understanding the biology."
Kelleher also noted that while tau has a large number of proteoforms, many proteins likely have far fewer.
"It's very different depending on the protein," he said. "Some proteins have a lot of this [proteoform] complexity, and certainly tau is one of them, but there are some that I have worked on that don't."
Given its role in a number of neurodegenerative diseases, tau and its proteoforms have received significant research attention. To date, Judith Steen, associate professor of neurobiology at Harvard Medical School, has conducted some of the most in-depth studies of tau proteoforms, using the mass spec-based FLEXITau approach developed by her lab. In a 2020 study in Cell, she and her colleagues mapped 95 posttranslational modifications on different tau proteoforms measured in post-mortem human brain tissue collected from individuals with Alzheimer's disease.
Other companies besides Nautilus have also delved into proteoform studies. During its recent investor day presentation, protein sequencing firm Quantum-Si highlighted the use of its platform for analyzing tau proteoforms. While the company's Platinum platform has largely been used to analyze peptides as opposed to intact proteins, it said it is enabling workflows using antibodies or other affinity reagents to detect posttranslational modifications on intact proteins, allowing for the detection of full-length proteoforms.
Mallick said that Nautilus came to explore tau through its collaboration with Genentech, noting that it was the first target it tackled with the drugmaker. He said Nautilus has explored proteins including p53 and EGFR through other partnerships and is considering additional targets in neurology and other areas.