NEW YORK – Over the course of the last decade, data-independent acquisition (DIA) mass spectrometry has gone from a cutting-edge approach used by a handful of labs to a commonplace proteomics technique.
Thermo Fisher Scientific's recent launch of its Orbitrap Astral instrument further cements DIA's place in the mainstream of proteomics as researchers now look to apply the method to new tasks like the analysis of post-translational modifications and other protein variants and grapple with looming data analysis challenges.
In its early years, mass spec-based proteomics was dominated by data-dependent acquisition (DDA) workflows, in which an 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, however, 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 in DDA experiments for MS/MS fragmentation and so are not detected. Additionally, because precursors are selected stochastically, the same proteins may not be measured in different experiments, which negatively impacts reproducibility.
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. Deconvoluting the complex spectra produced by fragmenting multiple precursors at once can be challenging, and traditionally DIA's depth of coverage has suffered as a result. In recent years, though, improvements in instrumentation and software have significantly boosted the performance of DIA methods.
Thermo Fisher's launch last month of its Orbitrap Astral adds a new instrument to the mix and marks the vendor's most serious effort yet to compete in the DIA space.
Thermo Fisher has long developed and offered DIA workflows for use on its Orbitrap-based instruments, but the Orbitrap technology has not been as natural a fit for DIA methods as have time-of-flight instruments sold by companies like Sciex and Bruker.
Michael MacCoss, professor of genome sciences at the University of Washington, who has developed a number of DIA methods for Orbitrap instruments, said that traditionally Orbitraps didn't offer enough speed and sensitivity to cycle through the full mass range of a sample with narrow enough m/z isolation windows.
"We did a lot of tricks like staggering the isolation windows" to improve the performance of DIA approaches on Orbitrap instruments, MacCoss said, noting that he and his colleagues were able with such techniques to get results comparable to those achieved on other platforms. Nonetheless, the fact that the Orbitrap technology was not an ideal match for DIA made the company hesitant to embrace the approach, he suggested.
Markus Ralser, director of the Institute of Biochemistry at Charité, agreed that the Orbitrap's attributes had influenced Thermo Fisher's attitude toward DIA.
"Their instruments are particularly good for data-dependent acquisition," he said. "They are known for creating very nice, mass accurate spectra. That is one of the strengths of the Orbitrap, but it is not so essential for data-independent acquisition. Data-independent acquisition profits a lot from high scan speeds, and TOF instruments have historically been faster."
Bradley Hart, senior director of analytical sciences marketing and life sciences mass spectrometry product marketing at Thermo Fisher, said the company had questions about whether DIA would see wide adoption.
"We sort of believed that DIA had some challenges; maybe it wasn't reproducible, the numbers were all over the place when it came to IDs, and quantitatively [it was] questionable," he said. This put the company somewhat out of step with many of its customers and the proteomics space more broadly as DIA workflows grew in popularity.
With the release of the Orbitrap Astral, which features an analyzer similar to a TOF in both speed and function, Thermo Fisher is making DIA a new point of emphasis. Given the company's prominence in proteomics, its embrace of the technique will likely drive further uptake, Ralser said.
"It's of course much easier for laboratories [using Thermo Fisher instruments] to incorporate DIA workflows into their thinking if their main provider of mass specs is now offering solutions for them," he said. "I think we will see a big shift because of this."
Joshua Coon, professor of biomolecular chemistry and chemistry at the University of Wisconsin-Madison, is a longtime Thermo Fisher mass spec user who has recently moved more heavily into DIA approaches. His lab has been running some DIA experiments using the company's Orbitrap Tribrid platforms but noted that the Orbitrap Astral appears particularly well suited to the technique.
"We have been a pretty heavy data-dependent lab until this last year, and for certain applications like plasma analysis, we have really begun to push on DIA," he said.
DIA has been an active area of method development with vendors and researchers developing a variety of new approaches in recent years.
Last year, Ralser and collaborators at Sciex developed the Zeno Swath DIA technique for use on the company's ZenoTOF 7600 instrument. The approach makes use of the instrument's Zeno trap to accumulate ions within a particular m/z window and then release them as the instrument cycles through that particular window, allowing for more efficient use of the instrument's measurement time.
Upon launch of the Orbitrap Astral at the American Society for Mass Spectrometry annual meeting in June, Thermo Fisher detailed a new DIA workflow for use on its instruments that combines its Vanquish NEO UHPLC system, µPAC NEO HPLC columns, EASY-Spray Nano Source, and Proteome Discoverer software with the Chimerys search algorithm.
Bruker has likewise been active in developing DIA workflows for its timsTOF platform. In 2020, a team led by the lab of Max Planck Institute of Biochemistry's Matthias Mann worked with the company to develop the diaPASEF method, which combines the parallel accumulation–serial fragmentation (PASEF) method previously developed by Mann's lab with DIA. The approach is similar to the Zeno Swath method in that it uses the timsTOF's trapping ion mobility spectrometry (TIMS) system to accumulate precursor ions and then release ions of a particular m/z window as the instrument is cycling through that window.
At this year's ASMS meeting, Bruker announced that it had licensed a new DIA technique called midia-PASEF (for maximum information-DIA) developed in the lab of Stefan Tenzer, head of the mass spectrometer core facility at University Medical Center of the Johannes-Gutenberg University Mainz. The approach, which Bruker plans to begin offering later this year on its new timsTOF Ultra instrument, uses ion mobility and overlapping quadrupole windows to provide what the researchers said is a 2.4-fold increase in sensitivity versus the conventional diaPASEF technique while also improving the ability to link the fragment ions produced during DIA runs back to their precursors.
This improving ability to link fragment ions back to their precursors is opening up new possibilities for DIA experiments, Ralser said, noting that it makes the approach more suitable for looking at protein post-translational modifications or features like splice variants.
Traditionally, DDA methods were better for such work "because they always gave you the precursor mass and the fragments," he said, which allowed researchers to assign PTMs or other detected features to the correct precursor ion.
Ralser noted, though, that new DIA methods like midia-PASEF as well as the Slice-PASEF and Scanning Swath approaches that he and collaborators have developed for use on, respectively, Bruker's and Sciex's TOF instruments, allow for more effective assignment of fragment ions to precursors, making DIA "as good for PTMs as DDA is."
He said that his lab has developed a new DIA approach it plans to release soon for the analysis of protein glycosylation.
A major challenge for DIA methods going forward will be handling the large amounts of data generated by these experiments, said Lukas Reiter, chief technology officer at Swiss proteomics company Biognosys. Reiter coordinated a meeting of the Data Independent Acquisition interest group at ASMS where he said the ability to scale up data analysis capacity was a common concern. The challenge, he noted, consists both of handling the large amounts of data produced by individual DIA experiments and the data generated by the large numbers of experiments that high-throughput DIA methods have enabled.
Reiter cited numbers he had put together showing that over the last decade, the throughput possible using DIA approaches has essentially doubled every two years, a trend that he said he expects will continue at least into the near future.
"A lot of people are struggling with the sheer amount of data," he said. "There was a lot of focus [at the interest group meeting] on how to analyze the data efficiently."
Data challenges aside, Reiter said the release of the Orbitrap Astral would likely push many researchers to move into DIA.
"Now we know that all three of the main manufacturers of proteomic mass specs are heavily investing in DIA-type hardware," Ralser said. "That gives a huge signal to the academic community and to all the little companies who develop tools and software that this is the way to go. I think it will create motivation to build new workflows and use these new capabilities because you see that this approach has come to stay."