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The Decade in Proteomics: Most Significant New Instrumentation


This is the second of four articles surveying leading proteomics researchers about the most notable achievements in proteomics during the 2010s. Part 1 can be found here, part 3 here, and part 4 here.

NEW YORK – Perhaps the best way to assess the advances in proteomic platforms over the last decade is to look back at the 2010 American Society for Mass Spectrometry meeting where vendors released what was, at the time, the cutting edge in instrumentation.

That conference saw the introduction of a mass spec system that would usher in one of the decade's major proteomic developments: the TripleTOF 5600 platform by AB Sciex (since renamed Sciex). At the 2011 ASMS meeting a year later, Sciex introduced its SWATH data acquisition workflow for the 5600, which went on to become the first widely used data-independent acquisition (DIA) mass spec method.

Michael MacCoss, professor of genome sciences at the University of Washington and a leader in DIA software and methods development, cited the emergence of DIA workflows as one of the key advances of the decade.

In traditional data-dependent acquisition (DDA) mass spec workflows, the instrument performs an initial scan of precursor ions entering the instrument and selects a sampling of those ions for fragmentation and generation of MS/MS spectra. Because instruments can't scan quickly enough to acquire all the precursors entering at a given moment, many ions — particularly low-abundance ones — are never selected for MS/MS fragmentation and so are not detected.

In DIA, on the other hand, the mass spec selects broad m/z windows and fragments all precursors in that window, allowing the machine to collect MS/MS spectra on all ions in a sample. Because the instrument collects data on all ions, the approach offers better reproducibility across samples than DDA approaches, though typically with a loss in depth of coverage.

MacCoss highlighted a key publication in the development of DIA approaches, a 2012 paper published in Molecular & Cellular Proteomics by ETH Zurich professor Ruedi Aebersold and colleagues that initially laid out the SWATH strategy.

Since then, vendors including Agilent, Bruker, and Thermo Fisher Scientific have all released their own DIA methods, and the approach has become increasingly popular, especially among researchers looking to do quantitative work across large numbers of samples. Waters has actually been offering a DIA workflow called MSE on its instruments since 2006, but it operates on different principles than the SWATH style methods that have predominated since the first was introduced in 2011.

MacCoss wasn't alone in making DIA his pick. Stefan Tenzer, professor of quantitative proteomics at Johannes Gutenberg University Mainz in Germany, also highlighted the emergence of DIA workflows. He and his colleagues have been developing new DIA workflows for Waters' instruments. 

Anthony Purcell, professor of biochemistry and molecular biology at Monash University in Australia, likewise said he considered "the maturation of data-independent acquisition strategies" to be the most significant development of the decade.

Stanford University professor Michael Snyder also highlighted DIA, observing that DIA workflows had made it so that "mass spectrometry can finally semi-quantify 5,000 proteins at high throughput."

Bernhard Küster, professor of proteomics and bioanalytics at the Technical University of Munich (TUM), cited DIA as well, but in a more qualified fashion, noting that "DIA is here to stay but [is] an interesting case as the community is divided over its merits."

"Everyone agrees that it is a great concept," he said, adding that some in the field have questions around issues like error estimation in DIA experiments.

Thermo Fisher's Q Exactive

The 2011 ASMS meeting also saw the launch of another technology that had a major impact throughout the decade — Thermo Fisher Scientific's Q Exactive mass spectrometer.

The Q Exactive instruments "doubled the size of global proteomic inventories, added high mass accuracy to peptide identifications, and enabled high-resolution targeted peptide quantitation [also known as parallel reaction monitoring]," said Daniel Liebler, president of mass spec services firm Protypia and formerly professor of biochemistry at Vanderbilt University School of Medicine. "They have been reliable and are distributed to many laboratories, so the quality of data across the field has been dramatically elevated."

Thermo Fisher released the Q Exactive, which combines a quadrupole for precursor selection with an Orbitrap mass analyzer, with the intention of competing for the first time in the Q-TOF market. While not a traditional Q-TOF, in that it uses an Orbitrap instead of a time-of-flight device for mass analysis, it was conceptually equivalent, and the instrument quickly proved popular for proteomics research and other applications, with Thermo Fisher reporting well over $100 million in revenues from sales of the platform in its first two years on the market.

In 2013, the company launched an updated version of the instrument, the Q Exactive Plus, and another update, the Q Exactive HF-X, in 2017.

Yuri Tsybin, founder of mass spec services firm Spectroswiss and formerly the director of the mass spectrometry service facility at the Swiss Federal Institute of Technology Lausanne, said that "instrument development was the key to many advances in proteomics" over the last ten years, adding that the Q Exactive in particular "was the key to proteomics progress in the past decade."

According to Tsybin,  the Q Exactive also lay the foundation for later Thermo Fisher instrument releases, like the Orbitrap Tribrid systems, which combine quadrupole, Orbitrap, and linear ion trap mass analyzer technologies.

TUM's Küster highlighted this instrument line as a major advance of the decade, calling the platforms "perhaps the most versatile [mass spec] instruments ever built."

The Q Exactive also proved Thermo Fisher's entry point into SWATH-style DIA mass spec, which was initially dominated by Sciex.

Ion mobility technology

Another frequently mentioned advance on the instrumentation side was the growth of ion mobility technology, which is now offered by all of the major life science mass spec vendors as a tool for upfront sample separation and is a key component of some recently launched high-profile platforms like Bruker's timsTOF Pro.

The University of Washington's MacCoss highlighted ion mobility along with his DIA pick. Scripps Research Institute professor John Yates III did as well, observing that "the last decade saw ion mobility devices start to have a real impact on proteomics. They started showing up on many new instruments and having a significant impact on instrument performance," he said.

"Ion mobility is finally on the move," said TUM's Küster.

Ion mobility uses differences in size, shape, and charge to separate ions in the gas phase. In proteomics, it is typically used to provide an additional layer of sample separation after conventional liquid chromatography. Researchers are also exploring whether the behavior of ions within an ion mobility system might provide additional data that could be used to improve peptide identification and quantitation.

Waters has been a leader in ion mobility, offering instruments featuring the technology prior to the last decade. Other vendors added ion mobility to their systems over the last 10 years, with, for instance, Sciex releasing its Selexion ion mobility technology in 2013 and Agilent making ion mobility available on its Q-TOFs in 2013.

More recently, Thermo Fisher in 2018 launched its new  High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) ion mobility system — a reboot of sorts for the company, which initially released a FAIMS device more than a decade ago but saw little uptake among researchers. Ion mobility is also at the core of Bruker's new flagship timsTOF Pro instrument, which offers a Trapping Ion Mobility System (TIMS).

And in the last years of the decade, new forms of ion mobility made their commercial debut, as both start-up Mobilion and Waters introduced ion mobility innovations.

Using SLIM (structures for lossless ion manipulation) ion mobility technology licensed from Pacific Northwest National Laboratory (PNNL), Mobilion partnered with Agilent to implement a new form of ion mobility on that company's mass specs, with a beta launched planned for this year and a broad commercial launch to follow in 2021.

The SLIM technology extends ion mobility path lengths beyond those allowed by conventional ion mobility spectrometry systems, potentially enabling much more extensive separations.

Waters introduced a conceptually similar technology last year as part of its new Select Series Cyclic IMS mass spec. The instrument features an ion mobility device with a circular path, which 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.

Richard Smith, a PNNL researcher and one of the developers of the SLIM technology, as well as a number of other ion mobility methods, said that he viewed the " the emergence and integration of different ion mobility separation technologies with mass spectrometry" as the most significant development of the last decade.

"The use of a second, and typically very high-speed, separation along with conventional liquid chromatography and mass spectrometry is now beginning to push proteomics into the realm of routine single-cell measurements, and making studies with many thousands of samples more practical," he said, adding that more advances were "to come as increasingly powerful ion mobility technologies are fully implemented and become more broadly available."

Amanda Paulovic, professor of oncology at the University of Washing School of Medicine and a researcher at the Fred Hutchinson Cancer Research Center, highlighted the improved multiplexing provided by the combination of advances in labeling reagents like Tandem Mass Tag (TMT) isobaric labels and instrument speed.

Küster likewise cited the development of higher multiplexed TMT labels, which now reach up to 16-plex and could enable reproducible quantification of proteomic samples at a depth of 8,000 to 10,000 proteins in around an hour.

George Mason University's Emanuel Petricoin highlighted the improvement of sample preparation techniques that have helped "both broaden proteomic coverage and increase throughput when coupled to [mass spec] or multiplexed assays [and] reach into the low-abundance range of the proteome in clinical samples — where all of the action is."

While most researcher selections were centered around mass spectrometry, a few highlighted other proteomic technologies.

For instance, Stanford's Snyder said that "capture agents for large-scale analysis finally reach reasonable numbers and scale," noting that proteomics firm Olink can analyze around 1,000 proteins with high throughput while Somalogic's current SomaScan platform measures around 5,000 proteins.

SISCAPA Assay Technologies CEO Leigh Anderson also suggested that non-mass-spec technologies have had a significant impact, noting that while he considered the publication of studies running 1,000-plus samples to be a major mark of progress, "most of these studies have been done with the SomaScan or Olink technologies," and that while "many things about [mass spec]-based proteomics have improved substantially, this has been steady progress rather than a milestone."

Robert Moritz, director of proteomics at the Institute for Systems Biology, likewise noted these advances in affinity-based platforms and suggested that the current decade of proteomics might be less dominated by mass spec than the previous one.

"I … foresee a fundamental shift in analysis technologies begun in this decade," he said. "With the developments of plate-based affinity approaches for protein quantitation that are more sensitive, highly multiplexed, and rapid, these technologies will replace many efforts currently performed by mass spectrometry."