Proteomics Platform Reduces Quantification to Counting

Nautilus Biotechnology introduces proteome-scale single-molecule protein identification using multi-affinity protein-binding reagents

Despite their sophistication and power, traditional proteomics technologies have failed to provide comprehensive coverage. For example, with conventional mass spectrometry, a single experiment measures no more than 8% of the blood plasma proteome or 35% of the cellular proteome. The quantification of low-abundance proteins has proven to be especially difficult.

Comprehensive coverage may require a completely new approach. This possibility is being explored by Nautilus Biotechnology. The company is developing a new technology that measures the entire proteome by counting each single, intact protein molecule within a sample.

The technology, embodied by the Nautilus Proteome Analysis Platform, aims to capture that which eludes existing proteomics technologies. These include traditional bulk quantification techniques (such as mass spectrometry and immunoassays) that have been adapted toward detection of single proteins; sequential processes (such as Edman degradation) that can determine positional information from amino acids within peptides; and directional translocation of a peptide through a nanopore channel.

Parag Mallick
Parag Mallick, PhD

“Single-molecule particle platforms have a couple of distinctive characteristics,” says Parag Mallick, PhD, co-founder and chief scientist at Nautilus. “With a single-molecule-based approach, identification is quantification.” He suggests that the Nautilus platform makes it possible for scientists to simply count identifications. Furthermore, because single-molecule analysis is the height of sensitivity, scientists needn’t adjust instruments for intensity or brightness.

A new approach

The Nautilus Proteome Analysis Platform has three key elements: sample preparation to immobilize each protein, iterative measurement using a new class of affinity reagents that recognize very short epitopes, and quantitation using a machine learning framework.

The ability to measure very short epitopes and to do so many, many times “allows scientists to be really efficient,” Mallick asserts. Rather than using antibodies that are specific to individual proteins, the Nautilus Proteome Analysis Platform uses a series of less-specific reagents that, combined, identify the proteins faster. Mallick anticipates that the system, still in development, will be able to “uniquely identify every single protein in the proteome with about 100 affinity reagents instead of the tens of thousands that are needed today.”

The use of multiple reagents and machine learning helps ensure accurate identification. “Ultimately,” Mallick explains, “you’re getting so much information from dozens of touches that misidentification becomes vanishingly improbable.”

The Nautilus Proteome Analysis Platform isn’t intended to replace other proteomics methods but to become a tool in the proteomics toolbox. By answering straightforward profiling questions, it can, Mallick says, provide a “deep profile of what’s there and how much.” That makes it useful for biomarker discovery, target discovery, and mechanism of action studies.

“The platform also can be used in a targeted mode,” Mallick remarks. The system leaves the protein intact, so researchers can analyze multiple versions (proteoforms) of a given protein. According to Mallick, this ability could help researchers “look at the proteome in detail” and help them “develop biomarkers that are substantially more specific.”

Proteomics for all

“I’ve been in the mass spectrometry proteomics community for decades,” Mallick states, “and I trained with Ruedi Aebersold, one of the fathers of the field.” Indeed, Aebersold was one of the founders of the Institute for Systems Biology in Seattle. Mallick relates that Aebersold inspired him to pursue the goal of making proteomics tools accessible for every scientist who wants them.

The idea, Mallick explains, is to measure the entire proteome in a single experiment “in a way that is scalable, robust, and easy.” Doing so, he adds, might “bring [proteomics tools] much closer to parity with genomics tools.”

“In 2016, when Nautilus was founded, measuring the total proteome in a single experiment was unachievable,” Mallick points out. At that time, the company faced two key challenges: the challenge of generating a super-dense single-molecule protein array, and the challenge of building affinity reagents to identify short epitopes. Understandably, the scientific community was skeptical.

Sujal Patel
Sujal Patel

When Mallick was still planning the company he knew that it would need more than “innovative technical foundations.” It would also need business expertise and a capacity for “amazing execution.” So, he reached out to Sujal Patel, who had co-founded and served as CEO of Isilon Systems (a company that had sold for $2.25 billion in 2010). When Mallick approached Patel about Nautilus, the two scientists had known each other for a decade. “Sujal dragged me to a white board for about 12 hours and grilled me,” Mallick recalls. “I left that meeting with a seasoned partner and co-founder—and the first check.” (Today, Patel is Nautilus’s CEO.) Farzad (Zod) Nazem, former chief technology officer at Yahoo, came on board as an angel investor.

Commercialization in 2024

Over the past seven years, those initial whiteboard challenges were solved. “At the most recent Human Proteome Organization meeting,” Mallick reports, “we presented data showing we could probe and reprobe these individual molecules for 100 cycles.” Now Nautilus is focused on new challenges: shifting to product ideation and taking the company’s technology to commercial scale.

One step in that plan took Nautilus public in 2021, raising $345 million through a private investment in public equity deal. That brought the total capital raised between the company’s inception and the end of Q2 2023 to $450 million. According to Patel, $287 million of the capital raised remains on the company’s balance sheet (slightly more than 60% of the capital the company has ever raised is still available to invest). “The vast majority of those funds went into research, and a bit went toward keeping the lights on,” Patel continues. “Since we’ve gone public, the focus has been on development, getting the research ideas and processes into shape to form a product. Next year, we’ll start to shift toward commercialization.” The goal is to get the Nautilus Proteome Analysis Platform into the hands of researchers.

Nautilus Affinity Reagent Probes
Designed to comprehensively quantify the proteome, Nautilus Biotechnology’s platform involves repeatedly flowing fluorescent affinity reagent probes over an array of up to 10 billion tethered proteins. The probes bind to short, defined protein motifs, enabling binding events to be measured. Since samples are not degraded, they can be washed and probed repeatedly with a variety of unique affinity probes.

Getting it right

Critics may point out that Nautilus is seven years old and is just now preparing to launch its first product to a scientific community still skeptical of proteomics innovations. But Patel believes that Nautilus’s “truly disruptive” proteomics technology isn’t something that can be rushed. “We are taking the time to ensure that the first product we build is the right product,” he says. “It will work in any laboratory in the world, and it will be reliable and perform at the specifications we’ve defined.”

Beyond its business office in Seattle (where Patel lives) and its technical facilities in the San Francisco Bay area (where Mallick is based), Nautilus has expanded south to San Diego (where the company has 7,000 square feet of laboratory and office space). Having sites in these locations helps Nautilus access specialized talent.

According to Patel, each site has its advantages. Seattle has talent in software and management. The San Francisco Bay area, which includes Silicon Valley, has talent in chemistry, biology, biochemistry, nanofabrication, hardware instrument engineering, and optical technology. And San Diego has talent surrounding Illumina.

Nautilus’s sites occur along the earthquake faults that run along the West Coast. Of course, this alignment is coincidental, but one may fancy that it reflects how Nautilus sees itself: A source of pent-up energy that is bound to shake things up. Were Nautilus to instigate a seismic shift in proteomics, tremors would certainly be felt by the company’s current collaborators, which include the Translational Genomics Research Institute, Genentech, Amgen, and the MD Anderson Cancer Clinic. Doubtless reverberations would be felt even more widely.