Single-molecule proteomics platform reveals biology’s hidden players

The ‘dark proteome’, a vast collection of proteins and proteoforms which remain largely uncharacterized, is one of the most significant blind spots in biology today. Although it remains largely invisible, due to gaps in current analytical methods and a historical focus on a small number of highly studied proteins, the dark proteome is thought to play a crucial role in cellular function. Now, a breakthrough platform from Nautilus™ Biotechnology promises to bring these hidden molecular players into the light. Employing Iterative Mapping for intact, single-molecule proteome analysis, this scalable platform is poised to overcome limitations in current methods, particularly mass spectrometry-based workflows, which dominate the field.

"In the cell, where you're talking about tens of thousands of different players or with proteoforms, any variant of a genetically encoded protein, you have millions of different entities potentially," explains Prof. Parag Mallick, co-founder and Chief Scientist at Nautilus™ Biotechnology and Associate Professor at Stanford University. "And not having access to that means there might be really important critical players that we simply have no idea what they're doing."

What we are missing in the dark proteome

Mass spectrometry, the current gold standard for proteome analysis, is limited by the physical constraints of its analytical process and is unable to offer a comprehensive look at the entire proteome. To illustrate the impact of current measurement gaps, Mallick offers a compelling analogy, "Let's say you had a set of friends over for a dinner party on Friday night, and you wanted to make a video recording of what was going on at that dinner party. But unfortunately, only half the people in the room showed up on the recording. How accurate is your understanding of what happens at the party going to be? It's going to be a challenge."

Undoubtedly the biological significance of the dark proteome cannot be overstated, with many proteins existing in low abundance and in only a handful of copies per cell, despite controlling a vast amount of cellular function. "Just because they're low in abundance, it doesn't mean that they're simple proteins." Mallick emphasizes. The dark proteome is a critical knowledge gap, with researchers missing key molecules as potential players in networks that drive cellular behavior, disease progression and therapeutic response. This leaves entire aspects of protein biology unexplored until there is technology available that can consistently measure these proteins with high quality, accuracy and reproducibility.

Single molecules, massive arrays

The Nautilus™ Proteome Analysis Platform addresses the limitations of traditional technologies using a method called “Iterative Mapping.” This is a fundamentally different approach that operates at the single-molecule level. Rather than digesting proteins and analyzing fragments, this platform immobilizes intact protein molecules on hyperdense arrays for non-destructive analysis¹.

"What we're doing is we are taking every molecule from the sample and gluing it down to one coordinate on that giant chessboard," Mallick describes. "And when I say giant, I mean truly giant, as our chessboard doesn't have 64 squares. It has billions."

This approach offers inherent advantages in sensitivity and dynamic range over traditional technologies. "By definition you cannot be more sensitive than a single-molecule counter. But dynamic range comes from measuring a large number of molecules," Mallick explains.

The platform applies Iterative Mapping in two ways:

• Iterative Mapping of proteoforms for detailed proteoform characterization.
• Iterative Mapping of proteins for comprehensive proteome coverage.

The primary differentiator is the probes used to analyze the single protein molecules.

For proteoform analysis, the platform uses highly specific affinity reagents to interrogate the modifications and alterations found on proteins. For broadscale analysis, it is designed to employ hundreds of probes that bind short protein sequences (~3 amino acids in length) that, through iterative cycles, build detailed molecular maps capable of identifying thousands of different proteins².

The process for both involves multiple rounds of fluorescent labeling, imaging, and washing, allowing researchers to ask sequential questions of each immobilized molecule. "That ability to iteratively ask a series of questions of each molecule is really the heart of the platform," Mallick notes. Ultimately, the platform provides quantification of substantively every protein in the proteome and in-depth views of specific proteins build detailed molecular maps of substantively the entire proteome.

Expanding the proteomics toolkit

Mallick does not envision this technology as a replacement for existing methods, but rather an essential addition to the proteomics toolkit. As he says, "We're not trying to kill mass spectrometry. We're just trying to create more options in people's toolbox. The way that I like to think about it is that the Western Blot did not kill the Coomassie Gel."

Accessibility is also a key design principle for the platform. With researchers at all stages of experience being able to access the platform equally, this allows researchers, who may not have extensive mass spectrometry experience, to undertake advanced proteomics analysis.

The platform's ease of use is remarkable for such sophisticated technology. It is designed such that "You literally walk up to it, drop a flow cell on, and press go. What comes out the other end is a spreadsheet that says, hey, here are the different proteins or proteoforms, and here's how much of each one is present," Mallick explains.

The future of protein analysis and drug development

Mallick envisions a future where researchers routinely combine broadscale proteome analysis with targeted proteoform studies. "What I anticipate is that many people will actually do both. They will want to understand the landscape of the whole proteome but then they'll also have a protein that they're particularly interested in."

He also considers the platform to be particularly well suited to artificial intelligence applications. With the reduction in missed data and minimized stochastic biases, the platform can enable more robust computational modeling of protein networks and cellular systems.

This will have broader implications for both drug discovery and biomarker identification. "By enabling access to this level of proteome and proteoform analysis, our hope is we could improve our ability to understand the specificity of therapeutics, and to understand potential drug targets that are truly present in disease tissues and not in healthy tissues, which would give us an improved view of therapeutic windows," Mallick explains.

Bringing the proteome to everyone

The ultimate goal remains clear. As Mallick emphasizes, "We want to get the proteome into everyone's hands." The dark proteome remains a challenge and an opportunity, with emerging technologies like the Nautilus™ Proteome Analysis Platform providing exciting promise. The journey from the dark proteome to the illuminated proteome is just beginning, and the potential for transformative discoveries has never been greater. With methods like Iterative Mapping that can detect, quantify and characterize proteins at a single-molecule level, we are getting one step closer to a complete picture of biology.

References

• Aksel, T., Qian, H., Hao, P., Indermuhle, P.F., Inman, C., Paul, S., Chen, K., Seghers, R., Robinson, J.K., De Garate, M., Nortman, B., Tan, J., Hendricks, S., Sankar, S. and Mallick, P., 2022. High-density and scalable protein arrays for single-molecule proteomic studies. bioRxiv. https://doi.org/10.1101/2022.05.02.490328
• Egertson, J.D., DiPasquo, D., Killeen, A., Lobanov, V., Patel, S. and Mallick, P., 2021. A theoretical framework for proteome-scale single-molecule protein identification using multi-affinity protein binding reagents. bioRxiv. https://doi.org/10.1101/2021.10.11.463967
• Joly, J., Budamagunta, V. and Zhang, Z. et al., 2025. Development of a method for large-scale single-molecule analysis of tau proteoforms. bioRxiv. https://doi.org/10.1101/2025.06.26.660445