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In 2019, Gains in Throughput Position Proteomics for Future Clinical Impact

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NEW YORK – For proteomics in 2019, the key word was throughput.

After two-plus decades spent struggling to drive experiment run times down to more manageable levels, a sense emerged this year within the field that this challenge had at last been met.

At a conference nine years ago, SISCAPA Assay Technologies CEO Leigh Anderson bemoaned the fact that, despite the thousands of papers published by proteomics researchers, he didn't "know of a single one in which anyone has actually run 1,000 samples."

Five years ago, a group of researchers managing to put together a mass spec platform capable of running as many as 1,000 isobarically labeled samples in 15 weeks was a newsworthy event.

This year, Thermo Fisher Scientific introduced an isobaric labeling approach developed by Harvard University professor Steven Gygi that could analyze the same number of samples in around half that time, and at depths of 8,000 to 10,000 proteins quantified per sample.

For shallower analyses, throughput has increased even more dramatically. For instance, researchers at the Francis Crick Institute and the University of Cambridge recently introduced a new software package for data-independent acquisition (DIA) proteomics that they said would allow users to quantify several hundred proteins in five-minute DIA analyses of undepleted plasma.

At this year's American Society for Mass Spectrometry annual meeting, Thermo Fisher presented data from an experiment in which researchers using the company's new Orbitrap Exploris 480 instrument quantified 3,000 proteins in a five-minute DIA run. At the same meeting, Bruker highlighted the throughput improvements provided by its timsTOF Pro instrument, with Vicki Wysocki, professor of chemistry and biochemistry at Ohio State University, noting in a presentation that with the instrument her lab was able to obtain the same number of protein identifications from an unfractionated 200-nanogram sample run over two hours of instrument time as they had previously obtained analyzing 12 micrograms of fractionated sample over 12 hours of instrument time.

Bruker has also presented a workflow combining the Evosep One LC system with the timsTOF Pro to quantify 500 proteins in depleted plasma at a throughput of 11.5 minutes per sample.

"You can now think about doing several thousand samples [in an experiment], which was [previously] not realistically achievable," said Oliver Rinner, CEO of Swiss proteomics firm Biognosys.

The hope, Rinner noted, is that the increase in throughput will allow proteomics to fulfill its long-awaited clinical promise. The throughput improvements reflect instrument and workflow advances, of course, but they also stem from a shift within proteomics in recent years from an emphasis on identifying and quantifying as many proteins in a samples as possible to experiments focused on achieving the throughput needed to obtain meaningful statistics about the clinical validity of potential protein biomarkers.

Echoing Anderson's comments from nearly a decade ago, Max Planck Institute of Biochemistry researcher Matthias Mann two years ago highlighted the need for such a shift in thinking, observing in a review in Molecular Systems Biology that improvements in mass spec technology — especially in terms of throughput — were making new and potentially more promising approaches to protein biomarker discovery more feasible and arguing for what he termed a "rectangular" plasma protein biomarker development strategy, measuring on the order of thousands of proteins in large patient cohorts in both the discovery and validation phases of a biomarker project.

"It is true that as everyone says, most early-stage [protein] biomarker studies are underpowered in terms of statistics," Rinner said. "It was very hard to do large-scale studies, and I think this is something that can change. It you can run biomarker studies with 1,000 samples, then the chance that in a follow-up validation or verification stage the biomarkers will prove true is certainly much higher. So, in that sense technology will help to move biomarkers forward."

In April, Biognosys presented data at the Mass Spectrometry Applications to the Clinical Lab (MSACL) annual conference from a mock clinical trial on colorectal cancer done in collaboration with drug and diagnostics firm Roche that Axel Ducret, a principal scientist at Roche and one of the leaders of the effort, said demonstrated proteomics could be done in compliance with GCP guidelines and on a timeline suitable for clinical trial work.

He said the results showed that DIA mass spec offers highly reproducible label-free quantitation that can be run with relative high throughput and a depth of coverage that is roughly equivalent to that offered by RNA-seq.

Rinner said that these technical advances are coinciding with increasing demand for biomarker research driven by the rise of the immune-oncology field and the need for better tools for predicting patient response.

"There has always been an interest in biomarkers, but it's stronger now because people clearly see the application" in immune-oncology, he said.

Beyond the boost in throughput, 2019 was a big year for ion mobility, as mass spec vendors like Agilent and Waters with well-established IMS offerings invested in new technologies while relative newcomers like Thermo Fisher and Bruker continued to grow interest in their IMS options.

Waters launched its Select Series Cyclic IMS mass spec, which features an IMS device with a circular path that both reduces the instrument's footprint while also allowing researchers to cycle ions of interest through the IMS device multiple times to achieve higher resolution separations. James Langridge, director of advanced MS technologies at Waters, said he expected the boost in separating power would prove particularly useful for experiments looking at hard-to-separate species like studying protein isoforms with isobaric modifications.

The device is similar to ion mobility technology developed at the Pacific Northwest National Laboratory and licensed by Exton, Pennsylvania-based Mobilion Systems, which uses structures for lossless ion manipulations (SLIM) technology to extend ion mobility path lengths beyond that allowed by conventional IMS systems and to direct ions through a system multiple times. Mobilion closed a $14.5 million Series A round in November. In September, the company announced it was partnering with Agilent to implement its SLIM technology on Agilent's QTOF mass spectrometers.

Thermo Fisher's High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) device, meanwhile, saw uptake among researchers for applications including label-based quantitative proteomics experiments, while Bruker began exploring the use of the collisional cross section (CCS) data provided by its timsTOF Pro's IMS system to provide an additional dimension of information that could enable more reproducible and sensitive analyses.

Thermo Fisher also in 2019 refreshed its high-end Orbitrap portfolio, launching its Thermo Scientific Orbitrap Exploris 480 instrument and its Thermo Scientific Orbitrap Eclipse Tribrid mass spec, both of which industry observers expect will help the company further establish itself as the leader in LC-MS proteomics.

A number of leading labs have also shown interest in Bruker's timsTOF Pro, which competes with the Orbitrap in the proteomics space and is one of the most significant new mass spec technologies to enter the field in recent years. In a note to investors, Barclays Analyst Jack Meehan suggested that the next two to three years would be crucial to assessing "the long-term trajectory of the instrument."

SVB Leerink's Puneet Souda likewise highlighted in a note the timsTOF Pro as a key growth driver for Bruker in 2020 and years following. Souda was more circumspect regarding Waters' Select Series Cyclic IMS instrument, noting that industry experts were concerned about software to serve the instrument and that it might not offer a sufficiently different level of performance from the Thermo Fisher or Bruker instrument to take market share.

Single-cell proteomic technologies also saw continued growth with a number of new developments in what is becoming an increasingly crowded field.

Branford, Connecticut-based IsoPlexis closed a $25 million Series C round that it is using to launch several new single-cell protein assays as well as expand its operations across the US, Europe, and Asia.

Single-cell imaging firm Akoya Biosciences also closed a funding round, raising $50 million to fund development of its Codex and Phenoptics spatial omics platforms.

San Francisco-based Mission Bio announced in November that it has partnered with BioLegend to develop assays for simultaneous DNA analysis and protein detection in single cells.

And St. Louis, Missouri-based Canopy Biosciences this year purchased German cytometry firm Zellkraftwerk, giving the company a single-cell proteomic offering.

In November, Stanford University spin-out IonPath launched its secondary ion mass spectrometry-based MIBIscope, which, according to the company, allows for simultaneous analysis of up to 40 proteins at the single-cell level and across five orders of dynamic range.

The instrument is a direct competitor of Fluidigm's CyTOF mass cytometry technology, and prior to the launch Fluidigm sued IonPath in the US District Court for the Northern District of California for patent infringement and tortious interference with some of Fluidigm's customer relationships.

IONpath has filed a motion to dismiss the suit, and Fluidigm has requested the court deny that motion. IONPath has until Jan. 9, 2020 to reply in support of its motion.

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