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Emerging Proteomic Technologies Could Provide Competition for Mass Spec


NEW YORK – Since proteomics came into being as a research discipline some 20-plus years ago, mass spectrometry has dominated the field.

Indeed, advances in mass spec are impossible to disentangle from the development of proteomics as the technology was the first to enable the study of thousands of proteins at a time — analysis at the scale of a proteome.

It's unlikely that mass spec will cede its position as the essential proteomic technology in the near term, but the rise of new protein analysis techniques like nanopore sequencing, have some questioning whether it will remain the method of choice for most proteomic experiments a decade from now.

"Technology doesn't stand still," said Lennart Martens, group leader of the computational omics and systems biology group in the VIB-UGent Center for Medical Biotechnology and a leading figure in the development of informatics tools for mass spec-based proteomics. "And it is clear that effort and money is now being thrown at these [new approaches]."

"We've now come closer than ever to realistic attempts to sequencing proteins in parallel at the single molecule level, and if the sequencing world is our guide, we know that a lot can happen once such technology starts to take off," he added. "I don't think many [mass spec] people are worried about their jobs just yet, as it is very early days, but researchers in the field are talking about these developments quite a bit because these are reaching a critical phase."

One notable voice that recently joined this conversation is sequencing firm Oxford Nanopore. While the company's primary focus has been on nanopore-based sequencing of nucleic acids, it has been active in nanopore protein sequencing, as well, both internally and through the funding of external research efforts. By and large, it has been close-lipped regarding its plans for the space, but during a presentation at the Nanopore Community Meeting in December, Oxford Nanopore Chief Technology Officer Clive Brown provided details on the firm's work on protein sequencing.

The company has built a breadboard for a droplet-based protein sequencing system using an approach originally published on by University of Washington researcher Jeff Nivala and University of California, Santa Cruz researcher Mark Akeson (when Nivala was a graduate student in Akeson's lab). The method uses the protein ClpXP to unfold a target protein and translocate it through a nanopore. The translocation of the protein through the pore generates a signal that might, in theory, be used to identify the protein and perhaps even determine its amino acid sequence including the presence of post-translational modifications.

According to Brown, Oxford Nanopore is currently using a "slightly revised" version of this approach to analyze proteins on its droplet system.

"We are generating now significant numbers of fairly consistent protein [signals]," he said. The company is using these protein signals to train algorithms against purified protein libraries.

"We have the first glimpse, here, of a natural protein sequencer," he said. "What that does … is it gets rid of all the HPLC, all the separation, the mass spec, the fragmentation, the reconstruction. … You can imagine just sequencing natural proteins directly from biological systems."

Oxford Nanopore is one of several commercial outfits pursuing new single-molecule protein detection. In December, researchers from French startup DreamPore along with several academic collaborators published on a nanopore-based system that was capable of identifying 15 of the 20 proteinogenic amino acids, suggesting that nanopore-based protein sequencing could be feasible. Long term, the company aims to develop the technology for large-scale sequencing of proteins and peptides. Nearer term, it is developing the platform for targeted detection of a biomarker for brain cancer recurrence, working in collaboration with clinicians at Lariboisière Hospital in Paris.

Gregory Timp, a professor of electrical engineering and biological sciences at the University of Notre Dame, who has been exploring the use of synthetic nanopores and sub-nanopores (pores with sub-nanometer diameters) for protein detection, said that a number of semiconductor companies have approached him about fabricating such pores for biosensing purposes.

Timp, who said he could not name the companies due to non-disclosure agreements he had signed, said that these firms were interested in the potential of synthetic nanopores to compete with existing technologies for analyzing DNA and proteins along with other molecules like carbohydrates.

"I think the semiconductor industry has big plans," he said.

Not that the path to commercialization will necessarily be a smooth one. Cees Dekker is a professor at the Delft University of Technology, a leading center for single-molecule protein work. Two years ago he and his Delft colleague Chirlmin Joo launched a company called Bluemics to commercialize nanopore-based protein detection technology developed in their labs.

The company has since paused activities, however, after struggling to find investors, Dekker said. "It hasn't been a very smooth start. It is maybe still a little early."

On the other hand, Dekker said that over the last two years his lab has made significant strides in refining its technology, demonstrating, for instance, that they are able to identify post-translational modifications localized to specific spots on a peptide. He added that he and his collaborators are currently preparing a paper they plan to submit that demonstrates further advances in their ability to sequence peptides via nanopore.

In 2017, Dekker and Joo organized the first Single Molecule Protein Sequencing conference, which brought a number of the field's leading figures to Delft to share their work. A second conference was held in September of last year in Jerusalem, hosted by Amit Meller, professor of biomedical engineering at Technion – Israel Institute of Technology.

Meller and his team are working on an approach that uses plasmonic nanopores to, as he said, "focus the sample to a specific point and provide a very local and intense electromagnetic field at that point."

This allows the researchers to detect fluorescent signal from analytes translocating through the nanopore.

"We think that if we are able to [fluorescently] label three amino acids of proteins and thread them through the nanopore, we can identify the vast majority of the human proteome with high accuracy," he said.

Key to this method is the use of deep learning techniques to interpret the fluorescent signals produced by the labeled proteins as they pass through the nanopore, Meller said.

Fluorescent labeling is also key to the single-molecule protein detection approach being developed by Austin, Texas-based Erisyon, a spinout launched to commercialize research from the lab of Edward Marcotte, professor of molecular biosciences at the University of Texas.

Erisyon's approach blends elements of next-generation sequencing and mass spectrometry, using fluorescent labeling of specific amino acid residues on target peptides followed by Edman degradation of those peptides. By immobilizing the peptides on glass slides and using microscopy to measure decreases in fluorescence as the labeled amino acid residues are removed from these peptides via Edman degradation, they are able to obtain partial sequences of these molecules. They can then match these partial sequences to a reference database to make peptide and protein identifications.

In 2018, Marcotte and his colleagues published a study in Nature Biotechnology that found that by labeling two amino acids they could identify proteins in mixtures containing on the order of 1,000 different proteins.

Since then, Erisyon has been working to further develop the technology, including adding to the number of fluorescent labels with which it can label analytes. Talli Somekh, the company's co-founder and CEO, said that he expected it would first apply the technology to targeted analysis of proteins and then move from there to complex samples and perhaps ultimately single cell proteomics.

Technion's Meller said he envisioned a similar path for his and other nanopore technologies, noting that the ability to detect a handful of protein biomarkers that are difficult to measure with existing technologies could be key to driving the field forward.

"There are clinical states in which probing very accurately only five or ten proteins would make a huge change in that field," he said. "So, I think there is where we will see the first applications, and I think the timescale for that will be a couple of years."

San Diego-based start-up Encodia is also using a labeling and degradation-based approach, developing DNA tags to label amino acids on peptides and then degrading these peptides one amino acid at a time. Upon removal, the DNA tags can be sequenced using conventional NGS, allowing for read-out of the tagged peptide sequences.

The firm includes several veterans of the NGS world, including its CEO Mark Chee, a co-founder of Illumina; Kevin Gunderson, formerly the senior director, advanced research at Illumina, as well as that firm's founding scientist; and Michael Weiner, one of the inventors of 454 sequencing.

Chee declined to comment on the company's work.

How do large life science mass spec firms view all this activity? Intriguing, but not threatening, suggested Andreas Huhmer, global marketing director for mass spectrometry solutions at Thermo Fisher Scientific. (Among other mass spec manufacturers, Sciex declined to comment for this story, and Bruker did not respond to requests for comment.)

Huhmer said that Erisyon's technology was exciting in that it could potentially enable protein identification "at a very massive scale."

"I would say that the proof has been provided that it can work," he said, however, he added that it was not clear to him how it would be applied commercially.

"I can see the clinical space being interested in that, particularly if you are not trying to read the entire proteome but are just focused on a [small] number of analytes," he said.

Huhmer said he doubted, though, that these emerging technologies would cut into mass spec's portion of the proteomic market, particularly given the continued growth of that market.

"Will researchers complement [mass spec] with other techniques? I would think so, particularly if they provide additional value," he said. "I doubt, however, that this would lead to a shrinkage of the market somehow."

Additionally, Huhmer noted that mass spec could benefit from the ongoing shift in focus within proteomics away from simply identifying proteins in isolation toward measuring their secondary and tertiary characteristics, meaning how they exist in their native states and how they exist within protein complexes.

"When I talk to my customers, what I hear more often is that they are primarily interested in functional information," he said. "And that primary information comes from understanding the secondary and tertiary structures. So, I think that our internal efforts are very clearly focused around understanding function, and that typically starts with understanding structure."