NEW YORK – Protein sequencing firm Quantum-Si (QSI) is drawing interest from established figures in the mass spectrometry world who are exploring the company's technology for research into proteoforms, different variants of proteins.
The labs of Northwestern University researcher Neil Kelleher and University of Virginia investigator Gloria Sheynkman, for example, recently presented on their work using QSI's Platinum protein sequencer to explore protein regions that are difficult to characterize via mass spec.
The projects are still in their early stages but researchers from both labs said QSI's sequencing technology appeared to complement their mass spec-based efforts to identify and analyze certain proteoforms of interest.
QSI's Platinum sequencer uses readouts of amino acid-specific probes, or recognizers, via a semiconductor-based sensing device that measures the timing of emissions following the excitation of target molecules, enabling single-molecule sequencing of peptides, including post-translational modifications.
The company launched sales of the instrument at the end of 2022, though it said in November 2023 that it was pulling back its commercialization efforts and operating in a "controlled commercial launch" until early 2024. In its recent Q2 2024 earnings report, the firm posted $584,000 in product revenue from sales of the Platinum instrument and its consumables.
QSI does not currently have probes to all 20 amino acids, which limits the peptide sequences the Platinum can reliably analyze, but Sheynkman said her lab has found the instrument can successfully sequence certain peptides that are not amenable to analysis by mass spec.
Sheynkman's research focuses on identifying protein variants produced by genetic processes like alternative splicing. Typically, her lab uses long-read RNA sequencing to identify genetic variants of interest followed by bottom-up mass spec to identify the proteoforms produced by these variants.
This work faces the challenge — common in mass spec-based proteomics — that certain peptides of interest have physical and chemical properties that make them difficult to detect using mass spec.
"There is just a fundamental limit" to which peptides can be detected by mass spec, Sheynkman said, noting that in her work, she and her colleagues have identified at the genetic level splice variants with very strong links to different diseases, only to find that the peptides produced by these variants "are just clearly not mass spec-able."
QSI's Platinum platform offers a potential approach for detecting some of these peptides.
"The mode of detection is just completely different" from mass spec, Sheynkman said, which raises the possibility that the instrument may find success with peptides where mass spec failed.
In early work with the platform, which Sheynkman presented at the American Society for Mass Spectrometry (ASMS) annual meeting in June, she and her colleagues managed to sequence a number of peptide targets they had not been able to characterize using mass spec. She cited as one example an experiment in which they used the Platinum to distinguish between two isobaric peptide forms where one contained the amino acid leucine and the other isoleucine.
In mass spec experiments, the two peptides "are chromatographically eluting together and [produce] one co-isolated spectrum," making them very difficult to tell apart, she said. The Platinum, on the other hand, clearly differentiated the two amino acids, even though they company does not have distinct probes for isoleucine and leucine.
"The kinetic pattern of the [probe] binding was really distinct [for each]," Sheynkman said. "So we could distinguish the isobaric peptides."
She said that thus far, she and her colleagues have projected what peptides the Platinum will be most useful for based largely on what probes are available to what amino acids, but that they are interested in generating data that will enable a more empirical look at where the platform can be effective.
"I really want to see the hard results, because once we have that, we will have a better estimate, and maybe it will be apparent when someone has a particular target if it is going to be amenable to QSI," she said.
Her lab has drawn up "a long list of protein isoforms that are intractable to mass spec" that they plan to tackle using the Platinum system.
Kelleher's lab at Northwestern is likewise exploring how the Platinum might help characterize proteins and protein regions difficult to get at using mass spec.
"We're really interested in looking at the complementarity and see what we might learn about each technique when we use them on the same sample," said Michael Caldwell, scientific officer at Northwestern's Proteomics Center of Excellence, where Kelleher is director. "Every technique has its biases, its limitations, and we in the mass spec field have lived with those for so long that it is really interesting to take an orthogonal approach and really be reminded of some of those limitations while also learning about what these new, high-potential technologies can really do."
In their work with the Platinum, the Northwestern researchers have focused on the protein IL-6, which Caldwell said was a "nice testbed" as it is a "very reasonable size" and can be produced recombinantly and easily expressed in cell systems. They presented a poster detailing results from the effort at the June ASMS meeting.
He noted that with existing top-down mass spec methods, researchers can achieve up to 80 percent to 90 percent sequence coverage of particular proteins but there are still regions of interest where coverage is limited.
In some of those regions, the Platinum has been able to deliver "specific recognition events for each amino acid," suggesting it could provide a more complete characterization.
Another area where the Platinum proved useful was detecting the post-translational modification pyroglutamate, which Caldwell noted can be challenging to do using mass spec alone.
"We're still working on the right way to talk about these two data types when you put them next to each other," he said. "Is there value that we can bring from that single amino acid information into top down [data]? That's an area that we are interested to look at in IL-6 and in future collaborations."
Kelleher also suggested that top-down data might prove useful for users of Platinum and other single-molecule protein analysis tools that will come to market in the future.
"If QSI had a known set of proteoforms, they would be advantaged," he said. "Any sequencing company will confront, necessarily, the complexity of the proteome that we are trying to regularize [via top down mass spec].
"We think that detailed top-down data on particular [protein] systems can really inform and enable single-molecule approaches that may struggle in a de novo setting without reference proteoform data," Caldwell added.
"We're thinking about these technologies as kind of orthogonal and complementary," said Meredith Carpenter, head of scientific affairs at QSI. "This is one of the first studies we've done along these lines, looking at top-down [proteomics data] along with the QSI data type."
"We need to gather more data along these lines to be able to understand how to best position the two and integrate all the data together, but … we really see this as another tool in the toolkit to be able to access some of those regions [less amenable to mass spec analysis] and dive more deeply into those areas," she added.