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At ASMS, Vendors Focus on High-Throughput Mass Spec Workflows

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SAN DIEGO (GenomeWeb) – Major instrument releases were relatively sparse at the American Society for Mass Spectrometry annual meeting this week, as many vendors focused instead on the development of applications and workflows for their existing platforms.

With regard to proteomics, this was most obvious in the promotion by several companies of more robust, high-throughput workflows for clinical biomarker development.

Proteomic researchers have for years critiqued the field's reliance on relatively small biomarker discovery cohorts and an emphasis on depth of coverage at the expense of throughput and reproducibility. Vendors have recently moved to address these issues via new instruments and workflows aimed less at identifying the most proteins possible in a sample and more at consistently and rapidly quantifying a large, but not comprehensive, set of proteins across many samples.

At this year's ASMS, this trend blossomed into something of a general industry consensus, with most major mass spec firms making it a focal point of their presentations.

For instance, Sciex, which was perhaps the first mass spec firm to formalize this idea when it announced several years ago a new emphasis on "industrial proteomics," had no major instrument releases this week but highlighted several of the initiatives falling under that industrial proteomics umbrella, including an ongoing effort by the Australian Cancer Research Foundation International Centre for the Proteome of Cancer (ProCan) to generate proteomic profiles of roughly 70,000 tumor samples, as well as work at the University of Manchester's Stoller Biomarker Discovery Centre running thousands of samples to discover biomarkers for diseases ranging from lung cancer to lupus.

According to Dominic Gostick, vice president and general manager of Sciex's LC/MS business, the company's current pipeline, which combines automated sample handling on a Biomek liquid handler with Swath mass spec on the TripleTOF 6600 instrument, is capable of running around 150 samples per week and can quantify around 4,000 proteins per hour.

Presenting during Sciex's ASMS press event on her research into biomarkers for cardiovascular disease, Jennifer Van Eyk, director of the Advanced Clinical Biosystems Institute at Cedars Sinai Medical Center, said the goal of such high-throughput workflows is to ultimately "merge discovery and validation."

With this, she echoed an observation by Max Planck Institute of Biochemistry researcher Matthias Mann who, in a review last year in Molecular Systems Biology, argued 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.

Speaking during Bruker's press event, Rohan Thakur, executive vice president in charge of the company's global life sciences business, highlighted Mann's review, using it to illustrate the company's ambitions for its timsTOF Pro mass spectrometer, which it launched at last year's annual meeting of the Human Proteome Organization.

The instrument's trapped ion mobility spectrometry (TIMS) technology enables the PASEF (Parallel Accumulation - Serial Fragmentation) mass spec method developed by Bruker and Mann's lab, which combines collection of ions via TIMS with rapid quadrupole switching on a QTOF instrument to enable the fragmentation of multiple simultaneously eluting precursor ions.

In a presentation at HUPO last year, Mann showed data from his group's experiments running the method on the timsTOF Pro. In a single-shot experiment using a 30-minute LC gradient they identified roughly 4,000 proteins and 800 unique proteins per minute.

Last week, Mann's group published a BioRxiv preprint that Thakur said detailed the current capabilities of the timsTOF Pro and PASEF workflow. Using a 30 minute LC gradient, the researchers were able to identify an average of 3,649 proteins in 100-nanogram HeLa cell digests.

To further increase throughput, Mann and his colleagues combined the timsTOF Pro with the Evosep One LC system, which uses a preformed gradient to improve assay robustness and cut downtime between injections. Using a PASEF workflow featuring 5.6-minute gradients, the researchers identified an average of more than 1,100 proteins across 10 replicates from 50 nanograms of HeLa cell lysate at a level of throughput that would allow running as many as 200 samples per day.

Thermo Fisher Scientific is likewise collaborating with Evosep to implement the Evosep One system in its clinical proteomics research. The two firms announced this week a joint effort centered out of Thermo Fisher's Boston-based Precision Medicine Science Center (PMSC) to develop robust, high-throughput clinical proteomic methods.

Thermo Fisher announced the official opening of the PMSC this week, which, as described by Ken Miller, the company's vice president of omics marketing, reflects the field's shift in thinking around biomarker development.

He contrasted the PMSC's approach to that of Thermo Fisher's Biomarkers Research Initiatives in Mass Spectrometry (BRIMS) Center, which the PMSC is meant to replace.

"BRIMS was really about establishing workflows and ways to do really deep analysis of samples, so you could run healthy and you could run disease [samples] and see the difference and say, hey, these might be biomarkers," Miller said. "And BRIMS would then say, we are going to take those markers and set up targeted quantitation assays. "

"What has changed in the world is, now people want to do this on hundreds or thousands of samples," he said. "And we can't do the BRIMS workflow on thousands of samples. It takes too long, it's too complicated, and it simply isn't scalable. I think in precision medicine today, it is all about how we migrate into the clinic. So we basically want to take the same principles of accurate, deep profiling of samples, but in a way that is fast and robust and can be translated into the clinic."

Miller noted that improvements in instrumentation have played a role in the increased emphasis on high-throughput, large-scale proteomic studies, but, he added, shifts in thinking within the field have also been a key factor.

"The way the technology is being applied is different, and the goal of the exercise is different," he said. "It's not so much about the deep dive biomarker discovery experiment. Instead, there are a lot of these big studies where the intent is to run 1,000 patients."

This change in thinking was also evident in Water's expansion of its collaboration with proteomics firm Biognosys, which the companies announced this week. The marketing agreement now covers the use of Biognosys' Spectronaut Pulsar X software and PQ500 Reference peptide kit on Waters' Xevo G2-XS QTOF running the company's SONAR data- independent acquisition method.

According to Waters, the combined platform is able to quantify around 500 proteins in 15-minute mass spec runs with single-digit coefficients of variation, making it potentially well-suited to high-throughput clinical proteomic work.

James Langridge, director of health sciences at Waters, said that since introducing the SONAR method several years ago, the company had determined that its major advantage was less in delivering deep proteome coverage than in maintaining sufficient coverage at fast run times.

"It is not so much the total information content from a longer gradient [running SONAR], but it's the ability to use that shorter gradient where you can say, ok, I want to do this in 20 or 30 minutes," he said. Like his counterparts at the other major mass spec firms, Langridge noted that this sort of workflow is increasingly in demand.

"It is quite clear there are cases where the number of samples people are looking at is increasing" in order to obtain statistically significant results, he said. "We see that with our customers where they say, 'We want to look at a greater number of samples and we can't afford to have instruments sitting there for a two-hour separation.' People want to get it to 30 minutes or under."

For vendors, these sorts of demands mean increased attention not only to instrument performance but to developing and testing "standards and protocols so that a lab can set up its system and keep it up and running and delivering quality data 24/7," Miller said. "It's really about creation of these robust SOPs that allow customers to operate their workflows at scale."

To this point, the company highlighted a recent internal study it undertook where it used a standard proteomic method for 700 runs in 11 different labs around the world and produced data across those runs and labs that showed a 90 percent correlation.

On a similar note, Bruker's Thakur highlighted a portion of the recent PASEF study wherein Mann and his co-authors noted they were able to run the timsTOF Pro for 1.5 years without cleaning the inside of the instrument. This, Thakur suggested, indicated the system's robustness and suitability for large-scale work where instrument uptime is key.

To date, proteomic biomarker discovery has a rather uninspiring track record. Whether the shift to a new approach focused on the use of larger cohorts and higher-throughput workflows will improve this situation remains to be seen.

Gostick said that Sciex's research partners are beginning to see results from their "industrial proteomics" efforts, noting that after several years of "scaling up the number of cohorts these labs are putting through these systems, we are starting, I think, to see some interesting and powerful biomarker discovery workflows, and some of those discoveries are now moving towards validation studies." He declined, however, to name any specific discoveries.

In any case, Gostick and representatives of other vendorssuggested that mass spec technology is no longer the limiting factor for these experiments that it once was.

"I think the [technology] is largely there," Gostick said. "I think we're in better shape today than we've ever been."

New Releases

While ASMS featured few new product releases this year, a few vendors did launch instruments aimed at the life sciences mass spec market. Bruker and Thermo Fisher Scientific, for example, both released new mass specs focused on intact protein analysis for markets including biopharma and structural biology researchers.

Bruker launched its scimaX magnetic resonance mass spectrometer, a 20 million resolution FT-ICR instrument aimed at phenomics and intact protein analysis. The system features a smaller footprint than traditional FT-ICR instruments and a conduction cooling system that eliminates the need for liquid cryogens, simplifying its use and making it potentially attractive to a broader audience.

On the phenomics side, the instrument is designed for high-throughput experiments using flow injection analysis and, according to Bruker, is capable of running up to 200 samples per day in this mode. The system is also aimed at biopharma and other researchers doing work on native proteins, as well as protein complexes and protein-ligand binding. Thakur also suggested the instrument could prove useful for top-down proteomics research, an area where researchers have over the last decade moved largely from FT-ICR instruments to Orbitrap systems.

Thermo Fisher's new Q Exactive instrument, the Q Exactive UHMR, is also aimed at structural biologists and biopharma researchers doing native and intact protein analysis. The instrument extends the mass range of the Q Exactive line to 80,000 m/z and adds high mass quadrupole selection and high HCD fragmentation efficiency, making it better suited to intact protein analysis than the company's previous offerings.

Thermo Fisher also launched its Orbitrap ID-X Tribrid mass spec, a version of its Orbitrap Tribrid instrument aimed specifically at small molecule identification. The instrument combines a quadrupole, Orbitrap, and linear ion trap along with an automated acquisition workflow, AcquireX, that the company said will help researchers go deeper into metabolomic samples by directing the instrument's analysis time toward unidentified molecules across series of runs.

Additionally, Thermo Fisher introduced a new triple quadrupole instrument, the TSQ Fortis, which is intended for routine analysis in areas like food and environmental testing.

Also of note, Shimadzu entered the Q-TOF market this week with the launch of its LCMS-9030 system. According to the company, the instrument has resolution of 30,000, a mass accuracy of 1 part per million, and a top acquisition rate of 100 Hz.

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