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Plasma Proteomics Seeing Renewed Interest as Mass Spec Technology Advances

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NEW YORK – Instrument advances and new mass spec workflows are revitalizing interest in plasma proteomics, according to a number of scientists familiar with the field.

Plasma-based analysis has long been a mainstay of proteomics research, but the approach lost some luster after, despite initial promise, it failed to deliver much in the way of medically useful biomarkers.

While researchers have published thousands of papers detailing early-stage plasma protein biomarker discovery efforts, very few new plasma protein markers or panels have made it to the clinic, and of those that have, most have enjoyed modest commercial success at best.

"I think there was a lot of hype many years back," said Emma Lundberg, associate professor at Stockholm's KTH Royal Institute of Technology. "People thought, we can do proteomics in plasma and this is how we're going to get into the clinic."

"And then, you know, as with many things that are hyped, it reached a point where you actually have to make things more robust and detect lower-abundance proteins and so on," she said.

As the tremendous interest in liquid biopsy applications within both genomics and proteomics demonstrates, blood is a very appealing sample source for clinical testing. It is both easily accessible and potentially highly informative as it contains molecules that could provide insight into all the organs and biological systems it interacts with as it moves throughout the body.

It is also, however, a very challenging sample to work with, especially in the case of molecules like proteins, for which, unlike nucleic acids, there is no method of amplification. This means that any assay must be able to measure proteins in plasma at their endogenous concentrations, a task that is made even more difficult by the plasma proteome's high dynamic range, which can span 10 orders of magnitude or more.

"I think proteomics has been really amazing in terms of studying the cellular proteome," said Jochen Schwenk, associate professor at KTH and director of the institute's plasma profiling facility. "But of course going into plasma, which is a particularly challenging type of sample, there was a need to [optimize] mass spec systems in a way that would let them cope with the particular complications of that sample."

Recent developments in sample prep and mass spec are driving an uptick of interest in the approach, he said.

Schwenk, who was senior author on a review of plasma proteomics published this month in the Journal of Proteome Research, cited as one example the BoxCar acquisition method developed by the lab of Max Planck Institute of Biochemistry researcher Matthias Mann, which uses the quadrupole of certain Thermo Fisher Scientific instruments like the Q Exactive or Orbitrap Fusion to improve analysis of low-abundance ions.

Applying the method to human plasma, the researchers found it provided an additional order of magnitude of dynamic range.

Schwenk suggested that another important development has been the rise of data-independent acquisition mass spec approaches and the demonstration that such experiments can generate interesting results in plasma without using extensive upfront fractionation. He highlighted a 2015 study by the lab of Swiss Federal Institute of Technology researcher Ruedi Aebersold in which the scientists used DIA mass spec to analyze plasma samples from the Twins UK Adult Twin Registry and investigate the sources of variation in protein expression. Using Swath-MS, the researchers quantified 342 proteins in 232 plasma samples.

"I think that study marked a sort of turning point where people said, ok, you can actually do a single-shot [plasma proteomic experiment]," Schwenk said.

Oliver Rinner, CEO of Swiss proteomics firm Biognosys, said that while in his view interest has always been strong in plasma proteomics, people were typically underwhelmed by the technique's capabilities.

"A few years ago, people would always be disappointed when I told them what you could get in plasma — the top 200 [most abundant] proteins or so, it's not very exciting," he said. "It's so much effort and so expensive, and the outcome really wasn't great."

On the other hand, Rinner said, "when you start to get up to 500 or 1,000, you start to see proteins that cover a much wider range of biological activities."

That is where, in terms of mass spec, at least, the field currently stands, he said, with the low end being for undepleted plasma and the upper limit more typical for depleted samples.

Like Schwenk, Rinner said the rise of DIA mass spec was an important factor in improving plasma-based experiments. He noted that the stochastic nature of data-dependent acquisition mass spec makes it a particularly poor fit for plasma, which is dominated by a handful of especially high-abundance proteins. Because DIA mass spec doesn't select ions to fragment stochastically but instead fragments all the ions in a sample it is better able to deal with the imbalanced nature of the plasma proteome.

"DIA is kind of the perfect fit to get deeper [in the plasma proteome] because now you can ignore the high-abundance proteins," he said. "It still hurts you in the sense of the [mass spec] detector sensitivity, but you can better target the low-abundance proteins."

Rinner said that mass spec-based proteomic assays can measure proteins in the low nanogram-per-milliliter range. He noted that this is not much of a change from previously, but the difference is that researchers are now able to measure many more proteins in that range.

"Before people would sometimes say they could see down to the nanograms-per-milliliter range, but that was just a single protein that for whatever reason was sticking out," he said. "Now we are able to see many more in that range."

Low-abundance proteins like cytokines "are still far out of reach," for untargeted plasma experiments using mass spec, he said, "but we are in the range where you can detect proteins that are shed from tissue, and more of them."

"If we can measure the top 500 to 1,000 plasma proteins, we get so much information that maybe we don't necessarily for all conditions need to look into the picogram-per-milliliter levels," Schwenk said. "Adding those [low-abundance proteins] that are very difficult to reach may be very informative in some cases, but if you want to go for a global-scale analysis maybe that is not necessarily needed."

Rinner said that the other main factor driving improvements in plasma proteomics has been better chromatography. Schwenk echoed this, citing in particular the LC firm Evosep as a notable player in this respect.

In a 2018 interview, Philipp Geyer, a postdoctoral researcher in the group of Max Planck's Mann (an indirect investor in Evosep), noted that he and his colleagues were using the company's LC systems for high-throughput plasma analyses. He said that using the Evosep LC for DIA experiments on a Thermo Fisher Q Exactive HF-X, the researchers were able to quantify around 300 proteins in undepleted human plasma in 21 minutes.

While this isn't particularly deep coverage, these 300 proteins include around 60 US Food and Drug Administration-approved protein biomarkers, Geyer said, noting that this suggests potential clinical applications.

He added that even 300 proteins measured with high throughput like 60 samples a day could be useful in enabling larger-scale protein biomarker studies.

In June, Thermo Fisher and Evosep announced that they were partnering to develop high-throughput clinical research proteomics workflows for profiling large plasma sample cohorts using the Q Exactive HF-X and the Evosep One LC system.

Biognosys and Thermo Fisher announced at the same time the extension of an existing partnership to encompass the development of DIA workflows for quantifying proteins in plasma and other biological fluids. Previously, the partnership combined Thermo Fisher's intelligence-driven mass spec technology with Biognosys' PQ500 Plasma Quantitation kit, which enables rapid, targeted quantification of 500 plasma proteins. The two companies have also worked to implement the BoxCar mass spec workflow.

Bruker has similarly highlighted the capabilities of its timsTOF Pro instrument for plasma proteomics, presenting a workflow combining the Evosep One with the timsTOF and the PASEF mass spec method developed by the Mann lab to quantify 500 proteins in depleted plasma at a throughput of 11.5 minutes per sample.

SISCAPA Assay Technologies CEO Leigh Anderson said that he is seeing increased interest in affinity-based plasma proteomic platforms like Somalogic's SomaScan and Olink's proximity extension assay-based protein panels.

The SomaScan platform can measure around 5,000 proteins in plasma or other samples while Olink currently has assays to 1,161 proteins. Olink offers its protein panels as kits and on a fee-for-service basis. Somalogic has in recent years moved away from a fee-for-service business model and is now focused solely on research collaborations where it has access to all the proteomic and clinical data generated by a project. It is working to use this data to build its own pipeline of clinical tests for the SomaScan platform and launched its first seven tests last month.

Schwenk suggested that while groups like Aebersold's and Mann's have provided demonstrations of the potential of existing plasma proteomic techniques, such approaches have not yet become commonplace within proteomics research.

"I think you can really say that a methodologies has arrived when people other than like the top three or four labs in the world are capable of doing it," he said. "You have had these sort of [leading labs] showing what [plasma proteomics] is capable of, but for it to really land you need to have places like UK Biobank or other places with large cohorts say, ok, we want to [analyze] our whole cohort and do plasma proteomics in addition to genomics."

One example of a research site moving in this direction is the University of Manchester's Stoller Biomarker Discovery Centre, which, since launching in 2016, has used DIA mass spec (primarily Sciex's Swath method) to analyze what Anthony Whetton, the center's director, said was "thousands and thousands" of patient samples, many of them plasma. Recently, he and his colleagues analyzed around 600 plasma samples looking for kidney disease biomarkers over the course of a few weeks.

I think [wider adoption] is going to happen," Schwenk said. "I think we are just at the beginning of the wave."

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