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Vendor Competition for DIA Mass Spec Market Heats up with Release of New Methods, Machines

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BALTIMORE (GenomeWeb) – With the launch of a new crop of high-resolution instruments at the American Society for Mass Spectrometry annual conference here this week, vendor competition for a slice of the growing data-independent acquisition mass spec market is heating up.

In particular, Thermo Fisher Scientific made an aggressive entry into this arena, formally launching four different DIA workflows for use on platforms including the Q Exactive, the Orbitrap Fusion, and the Q Exactive HF, which the company introduced this week.

With these releases, Thermo Fisher has taken direct aim at AB Sciex's DIA workflow, Swath, which the company launched in 2011 for use on its TripleTOF 5600 instrument. In fact, Thermo Fisher has named its basic DIA workflow for the Q Exactive Swath DIA – whether as an homage or a provocation is unclear.

AB Sciex likewise introduced a new DIA workflow this week, Swath 2.0, an updated version of the original methodology, which played a significant role in driving the current interest in DIA techniques.

Traditionally, shotgun proteomics experiments have used data-dependent acquisition wherein the mass spectrometer performed an initial scan of precursor ions entering the instrument and selected 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 ions – 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.

Enabled by the increasing scan speed of linear ion-trap instruments, the technique was introduced in a 2004 Nature Methods paper by Scripps Institute researcher John Yates III – who implemented it on a ThermoElectron LTQ instrument. Since then, DIA techniques have been employed by a number of proteomics researchers using a variety of different platforms.

Waters introduced the first commercially available DIA methodology in 2006 with its MSE workflow. This approach differs from Swath and the similar methods released this week by Thermo Fisher, however, in that instead of cycling through broad m/z windows, the mass spec in MSE fragments the entire m/z range at once and then uses retention time and ion mobility data to match fragment ions with their precursors.

And so, upon its 2011 release of Swath – which the company developed in collaboration with Swiss Federal Institute of Technology Zurich researcher Ruedi Aebersold – AB Sciex essentially had the market for that DIA methodology to itself.

In particular, it had the leading solution for pulling targeted quantitative data out of Swath-style datasets. As University of Washington researcher Michael MacCoss told ProteoMonitor in a 2012 interview, while Swath-style DIA was possible on high-resolution instruments from a number of vendors, other vendors hadn't really "pushed a solution for looking at analytes in the data in a targeted way [like] AB Sciex" had.

This week, Thermo Fisher put forth its first formal challenges to AB Sciex's dominance in this area, officially launching four DIA workflows: Swath-DIA for use on the Q Exactive family of instruments including the newly released Q Exactive HF; pSMART DIA for the Q Exactive instruments; Msx-DIA for use on the Q Exactives; and WiSIM DIA for use on the Orbitrap Fusion.

The Swath-DIA works essentially the same way as AB Sciex's first version of Swath, cycling through the full mass range in 32 windows of 25 daltons each.

The pSMART-DIA, meanwhile, cycles through the full mass range in windows that vary depending on how crowded the spectrum is in a given part of the range. For instance, Ken Miller, Thermo Fisher's vice president of marketing for life sciences mass spectrometry, told ProteoMonitor this week, "in the crowded part of the spectrum we tend to do 5-[dalton] windows and when we get up to the higher m/z windows where there are fewer precursor ions, then we open it up to maybe 10-[dalton] windows.

The msx-DIA workflow, which was developed in collaboration with UW's MacCoss, uses the Q Exactive's combination of quadrupole, C-trap, and Orbitrap to collect data in 20-dalton chunks, each composed of two separate 10-dalton windows taken at random from different parts of the mass range. This multiplexing approach improves performance by combining the sampling frequency provided by large acquisition windows with the specificity and reduced ion interference of smaller windows.

The WiSIM DIA on the Orbitrap Fusion uses 200-dalton selected ion monitoring scans at 240,000 resolution to cover the sample mass range, while in parallel performing 17 sequential MS/MS scans at 12-dalton windows to cover the 200-dalton SIM windows. According to the company, the method offers up to four orders of dynamic range and attomole levels of quantitation.

AB Sciex's newly released Swath 2.0, meanwhile, provides four to four-and-a-half orders of dynamic range, said Mark Cafazzo, AB Sciex's global market manager for proteomics. In experiments with yeast lysate, company researchers have used the technique to quantify roughly 22,000 peptides with a 1 percent false discovery rate and coefficients of variation across replicates typically below 20 percent, he said.

Like Thermo Fisher's pSMART-DIA tool, Swath 2.0 uses variable window sizes to adjust for different portions of a sample's spectrum. "Ultimately what that results in is tighter quantitation," Cafazzo said.

Perhaps unsurprisingly, both Thermo Fisher and AB Sciex claim superiority for their systems and DIA methods. Broadly speaking, AB Sciex asserts that the higher speed of a QTOF instrument compared to an Orbitrap provides an inherent advantage for DIA analyses.

As Aaron Hudson, senior director of the academic and omics business at AB Sciex, argued this week, the TripleTOF 6600 scans 10 times faster than the new Orbitrap HF, meaning more data points across a peak to ensure good quantitation.

Thermo Fisher's Miller countered, however, that while QTOFslike the 6600 do have higher scan speeds, the lesser quality of QTOF data means such machines need more scans to obtain good data.

"To get reasonable spectral quality, QTOFs have to average a lot of scans, so their effective scan rate, once they do this spectral accumulation and averaging, is no faster than ours," he said.

Instead, the higher resolution and mass accuracy enabled by the Orbitrap provides such instruments with an advantage in terms of identifying and quantifying analytes within the complicated spectra generated by DIA approaches, Miller argued.

"If you take a 25-[dalton] window of a complex mixture, there is going to be a tremendous number of precursor ions in that window," he said. "And now you are going to fragment them all together to create this very complex composite MS spectrum. So when you go to decipher what is in that spectrum, it is our contention that having access to very high resolution and very high mass accuracy will allow you to resolve those components and identify them much more accurately than you can with QTOF data."

Waters, meanwhile, argued the superiority of its MSE DIA approach, which, rather than using an upfront quadrupole to cycle through mass windows, fragments an entire sample at once, using retention times and ion mobility data to then match precursor and fragment ions.

One of the primary advantages of that approach compared to Swath-style methods, said James Langridge, director of discovery, pharmaceutical, and life sciences at Waters, is that analyzing the entire mass range at once offers improved sensitivity.

"When you move to a Swath-type environment, you are moving the quadrupole and selecting the mass ranges, and so when you select one mass range, that means that everything else [outside that mass range] is being filtered out, so your sensitivity is lower," he said.

One point of support for this argument is a Nature Methods study published in December by researchers at the Johannes Gutenberg University Mainz, in which they used an MSE method termed USMSE that incorporates ion mobility drift times to improve precursor fragmentation efficiency.

Applying the approach to an analysis of HeLa cell lysate on a Waters Synapt G2-S instrument, the team identified 36,551 peptides and 3,795 proteins with a 90-minute nanoLC gradient. Notably, given the traditional superiority of DDA methods for making peptide IDs, this surpassed the performance of a comparable DDA analysis on a Thermo Fisher Q Exactive.

Langridge said that Waters had not yet repeated the experiment on its new Xevo G2-XS instrument but plans to incorporate the workflow into its instruments.

Bruker Vice President for Life Sciences Research and Proteomics Ole Vorm told ProteoMonitor, however, that despite the excitement surrounding DIA methods, he felt conventional DDA would remain the method of choice – particularly as DDA methods continue to improve.

DDA typically provides better coverage in terms of protein IDs, but this coverage is more variable due to the fact that what ions are observed depends on which ones happen to be selected for fragmentation during a run.

DIA, on the other hand, tends to identify and quantify a more consistent – if smaller – set of proteins across multiple runs, giving it a potential advantage for comparing protein levels across samples.

Conventional DDA analysis "will always remain," AB Sciex's Cafazzo said, but, he added, the demand for high-quality quantitative data was driving demand for DIA approaches like Swath.

"If you have replicates you want to compare, but on this set only three out of five replicates have data for the proteins you are interested in, then how can you say you are really measuring up and down regulation?" he said. "So the comprehensiveness is where people see the value in [DIA techniques.]"

Vorm suggested, though, that the performance of modern mass specs allowed DDA analyses to capture very consistent data across replicates.

"When we do DDA, there is always some variability, but we are down in the few percent," he said. "The vast majority of the [identified proteins] are the same."

Nonetheless, DIA proponents maintain that that method will typically provide better quantitative precision and accuracy over multiple samples.

As ETH Zurich's Aebersold told ProteoMonitor in a November interview, "when you do repeat analyses, which is what we are most interested in, there is a crossover point at which the somewhat smaller number of peptides [compared to DDA analyses] identified per Swath run [becomes] a higher number of consistently identified peptides."

He added that an analysis by targeted proteomics firm Biognosys – of which he is a co-founder – had determined this crossover point to be around three to four samples.

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