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Mass Spec Instrument Advances Are Cause for Optimism in Top-Down Proteomics

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NEW YORK – Due in large part to its technical complexity, top-down proteomics has remained a relatively small niche within proteomics research.

Recent developments in mass spectrometry could herald an uptick in interest in the technique, however, suggested Neil Kelleher, professor of chemistry at Northwestern University and a leading top-down researcher.

"Is 2019 going to be a tipping point? I can't predict the future," he said. But, he added, new instrument offerings have the potential to expand top-down to a wider audience.

"There has been the perception of [top-down], where everyone has said, 'Oh, it's harder, there are no instruments, there are no commercial platforms,'" he said. "But that's now not at all correct."

Unlike conventional bottom-up proteomics, in which proteins are digested into peptides and then separated by liquid chromatography and analyzed by mass spec, top-down proteomics fragments and analyzes intact proteins. The approach offers potentially richer data on the different protein forms, or "proteoforms," in a sample, allowing researchers to more directly study the role of phenomena like post-translational modifications or sequence variants. However, analyzing intact proteins on a proteome-wide scale is more challenging than bottom-up approaches and top-down has lagged behind both in terms of the number of analytes it can measure and sample throughput and in the number of researchers using the technique.

Running down recent instrument advances, Kelleher cited in particular Thermo Fisher Scientific's recently launched Orbitrap Eclipse Tribrid, which features a number of upgrades targeting researchers doing top-down proteomics and intact protein analysis. He also singled out Water's BioAccord mass spec system, which the company launched at the beginning of the year.

While the BioAccord is intended primarily for monitoring applications in biopharma development, Kelleher said he thought the platform's accessibility could make it something of a gateway drug for top-down research.

"It's really robustly built and its relatively inexpensive and you could have a bachelors or masters [degreed] person run it, not a PhD proteomicist," he said. "I was very enthused by that because you don't need a real expert. And you want to democratize top-down, you want to get economies of scale kicking in, you want to get an ecosystem stood up."

"As soon as you have intact ions flying around in your instrument, that's the slippery slope," he said. "Once you have those intact ions flying around, you get people used to [the idea of top-down proteomics]."

The BioAccord, Kelleher acknowledged, has considerably lower performance than instruments typically used for top-down work, but he suggested that the platform shows a path for vendors to construct more user-friendly top-down devices.

In an email, Waters spokesperson Brian Murphy said that while labs "performing non-biopharma applications have inquired about the BioAccord system… they are in the minority."

Kelleher said that his lab would consider buying a BioAccord just to explore what it can do, given its high volume and ease of use.

Thermo Fisher's Eclipse is a more traditional top-down instrument, and while the system is pitched primarily to bottom-up researchers, a number of the new features it offers are particularly useful for intact protein work.

One such feature is its ability to do proton transfer charge reduction (PTCR), an ion-ion reaction that allows researchers to reduce the charge of peptides or proteins trapped in the instrument's linear ion trap.

This charge reduction helps simplify the often-complicated spectra generated during intact protein and top-down proteomic experiments, said Ken Miller, Thermo Fisher's vice president of marketing, life sciences mass spectrometry.

"This is a way to simplify and interpret spectra that were a major challenge and major limitation for this entire [intact research] area," he said. "It should be a significant advancement in terms of complex intact protein mixtures. People are talking about doing proteoform-level separations and characterizations now, and PTCR will be a big help there. We see this as being a real game changer for top-down and complex intact analysis."

Kelleher said his lab has been using the Eclipse since the beginning of the year and that it plans to formally acquire one later this year. He said his lab is still using previous Orbitrap instruments including the Fusion Lumos and Q Exactive for much of its top-down work but that "for our major biomarker discovery and top-down proteomics projects, [the Eclipse] is what we will use when we want top, top-of-the-line [performance]."

The instrument offers around a four-fold improvement in speed for top-down, Kelleher said, noting that it also allows for analysis of either native or denatured whole proteins. The ability to look at native proteins (proteins that are still structurally intact) is potentially significant in that top-down proteomics has difficulty with large denatured proteins (above 50 kiloDaltons), whereas this is a less of an issue with native proteins, he said.

Beyond the BioAccord and the Eclipse, Kelleher suggested Bruker's timsTOF Pro also interested him as a potential top-down device, though the company has primarily targeted that instrument at bottom-up researchers. Top-down and intact protein researchers have commonly used Bruker's FT-ICR instruments like its Solarix and Scimax systems, though they have also used QTOFs like its MaXis II and Impact II platforms.

Lloyd Smith, a professor of chemistry at the University of Wisconsin-Madison, noted that the impact of Orbitrap technology on top-down research has been significant but is tapering, though he said that he was not at the recent American Society for Mass Spectrometry annual meeting where the Eclipse was released.

"The Orbitrap made a huge difference in taking very sensitive, fast, high-resolution mass spec and making it accessible to lots of people," he said. "I wouldn't be doing the work that I'm doing without the Orbitrap, and that's because I didn't really want to invest into the FT-ICR business. So there has been a huge jump in the field because of that."

That said, Smith noted that he believed "the Orbitrap platform is now at a slower stage of growth and development as a platform, which is kind of natural. It's more, sort of, 20 percent better here, 40 percent better there."

While mass spec advances have improved the capabilities of top-down approaches and driven increased uptake, Smith suggested the field still remains far from the ultimate goal of enabling the routine measurement of tens of thousands of proteoforms.

"I believe that this is critical to biology," he said. "To understand how biochemical systems work you need to know the molecular players in detail. You need to know how these molecules are being changed in response to perturbations. Right now that system-wide proteoform-level information is largely unavailable."

Kelleher agreed that despite his optimism regarding the recent wave of new mass spec instruments, top-down based biomarker discovery is currently "a tough slog," and that it will take years and "tens of millions of dollars" to demonstrate the effectiveness of that approach.

In the meantime, though, he said more targeted studies could help make the case for an increased focus on top-down work. He cited work his lab has done look at the protein-level effects of mutations in the KRAS oncogene where they identified several mutation-linked post-translational modifications involved in KRAS activation and membrane attachment. 

Analyzing intact proteins allows researchers to "get at all the sources of variation that underlie your biology, your PTMS, isoforms, and variants," Kelleher said. "But you have to go through the proteoforms to get to all the goodies that you want."

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