NEW YORK – Since it was launched by Sciex eight years ago, Swath and Swath-style workflows have dominated data-independent acquisition (DIA) mass spec.
The recent American Society for Mass Spectrometry annual meeting saw the release of several new DIA approaches that update and expand upon the method.
Perhaps most notable was Bruker's introduction of a new DIA technique, called diaPASEF, which is short for data-independent acquisition combined with parallel accumulation-serial fragmentation.
The method leverages the upfront trapping ion mobility system of Bruker's timsTOF Pro instrument. While Bruker's QTOF instruments have been capable of performing DIA experiments, the approach has not been a point of emphasis for the company.
Bruker indicated during its ASMS press meeting that it considers the diaPASEF method a key workflow for the timsTOF system, with Frank Laukien, the company's president and CEO, calling it "a very significant new technology" that would enable more sensitive and higher throughput proteomic experiments.
Devised by a team of researchers including the Max Planck Institute's Matthias Mann and the Swiss Federal Institute of Technology Zurich's Ruedi Aebersold, who developed the original Swath method, the diaPASEF approach uses the timsTOF Pro's ion mobility system to trap and release ions based on their ion mobility characteristics, which allows the instrument to sample a higher percentage of ions than is possible in a conventional Swath experiment.
Traditional Swath approaches use an instrument's quadrupole to select all the ions in a broad m/z window for fragmentation. The instrument steps sequentially through these windows until it has sampled the full m/z range being analyzed.
While the instrument is focused on one m/z window, however, it is discarding all the ions that fall outside this window, which means they are effectively wasted, lowering the sensitivity of the experiment. The diaPASEF approach address this issue by using the timsTOF Pro's trapping ion mobility device (TIMS) to capture the ions before they are selected for by the instrument's quadrupole. Once trapped in the TIMS device they can be released based on their ion mobility characteristics, which are closely related to their m/z. This allows researchers to coordinate release of ions from the TIMS device with the stepping of the quadrupole through the m/z range being analyzed, which significantly reduces the amount of wasted ions.
"You are accumulating much more signal and sampling many more ions," said Florian Meier, a postdoc in Mann's lab and first author on a bioRxiv preprint presenting the method. "So you would expect to see more complete data and better quantitative accuracy."
"The fact that you are trapping all the ions and then release[ing] them based on their size, combined with the speed of [the timsTOF instrument], allows you to be much more efficient with the use of the ions," said Michael MacCoss, a professor of genome sciences at the University of Washington and expert in DIA and other quantitative proteomics methods.
MacCoss, who was not involved in developing the diaPASEF method, said that while the method has promise, he is interested to see more extensive data on its quantitative performance.
"Does it have the precision, the dynamic range, of other [approaches]?" he said. "From my perspective that is still yet to be shown."
Meier noted that work on the method is still in the early days and the results reported in the preprint are subject to change, but said that the results from that work appear promising. Running a 200-ng HeLa cell sample in triplicate using a two-hour LC gradient, the researchers quantified 6,974 proteins, 96 percent of which they quantified in all three samples, with median coefficients of variation of 11.5 percent.
The diaPASEF approach also has the potential to improve on traditional Swath-style methods by offering an additional parameter to help identify the ions analyzed in an experiment. Because DIA experiments fragment broad m/z windows simultaneously, they generate highly complex spectra from which researchers must extract information on individual peptides. Currently, DIA analyses extract this data based on precursor and product ion masses and LC retention times. The diaPASEF method, Meier noted, adds ion mobility information to that mix.
The extent to which this will prove helpful "is something we are still investigating," he said. "But it is additional information about the ion that we get for free with this experiment setup."
MacCoss agreed that this additional data point could help improve DIA analyses.
DIA "tends to be plagued by interference because you have these wide isolation windows," he said. "It's possible that ion mobility improves that specificity because it is an extra dimension."
However, an ion's mobility is "fairly well correlated with mass, so it's not like it's orthogonal [to existing parameters]," MacCoss noted. "So time will tell. What we need is lots of other people looking at the data and trying to make sense of it and to figure out the best ways to analyze it and interpret it.
Meier added that work needs to be done to optimize the precursor windows and other parameters used in the experiment.
"In classical DIA, you have precursor windows [based] only on the m/z dimension, but now we can use precursor windows based on m/z and ion mobility, and it is not trivial to figure out the best way to place these precursor windows" he said. "It is always a balance between the coverage of the precursor space, the sensitivity of the assay, and the total acquisition cycle time you would like to spend. So there are many parameters, and we just started testing them with a few standard samples, but there is a lot more research to do in order to understand the parameter space we can now adjust to."
The Mann lab was also behind another new DIA method presented at the 2019 ASMS meeting, this one based on the BoxCar acquisition technique it described last year in a paper in Nature Methods.
Developed for use on Thermo Fisher Scientific's Orbitrap instruments, the method adjusts the sampling of ions at the MS1 level during mass spec analysis to expand the instrument's dynamic range and sampling depth.
Shotgun proteomics experiments use an initial MS1 scan that measures the mass-to-charge ratio and signal strength of the peptides, and then an MS2 scan that analyzes the fragmentation pattern of the peptides, allowing researchers to match them to patterns in peptide databases and make identifications.
A major challenge for proteomics, particularly in experiments using complex samples with high dynamic ranges, is that only a small proportion of peptides in a sample are selected for analysis. This means that high-abundance peptides are overwhelmingly selected for, making identification and quantification of lower-abundance molecules challenging and highly variable across different samples.
The BoxCar method uses the upfront quadrupole of Orbitrap instruments like the Q Exactive or the Orbitrap Fusion to select ions from a wide variety of m/z ranges to pass on for analysis, diluting the presence of the high-abundance species and selecting a larger proportion of low-abundance peptides.
Applying the approach to a conventional data-dependent acquisition experiment, they found that it substantially improved the number of proteins quantified per run and the reproducibility of the proteins quantified across runs while providing an additional order of magnitude of dynamic range.
In work presented at ASMS, the Mann lab along with collaborators at Thermo Fisher and proteomics firm Biognosys adapted the technique to a DIA experiment, where the increased signal and expanded dynamic range at the MS1 level led to a boost in quantified proteins and an improvement in reproducibility compared to a conventional DIA analysis.
Sciex, which set the stage for DIA mass spec's widespread adoption with the release of its Swath DIA method in 2011, also released a new DIA technique at the ASMS meeting. Called Scanning Swath, the workflow uses a sliding quadrupole window as opposed to stepped isolation windows, which boosts performance by improving ion accumulation and providing additional information for matching of precursor and fragment ions.
The additional information for matching precursors and fragments comes from the fact that as the isolation window slides across the mass range of interest, precursor and product ions can be seen entering and leaving the window, and those entry and exit times provide another parameter that can be used to resolve complex spectra, said Christie Hunter, director of applications at Sciex.
She gave the example of a pair of co-eluting peptides that would be challenging to tease apart using a conventional Swath approach.
"If you look at that fourth dimension you can see that you can tell which fragment ions go with which parent ion, because they differ slightly in their [isolation window] entry and exit points," she said.
According to Hunter, Sciex researchers have managed a 30 percent boost in peptide identifications compared to conventional SWATH approaches. She added that she expected the method's performance will continue to improve as researchers optimize the algorithms and acquisition strategies employed.
"Because they have information about when the peptide enters the isolation window and when it disappears, they can actually [achieve] much higher precursor specificity than they could based on the isolation width of their [quadrupole] alone," MacCoss said, noting that the approach was conceptually similar to one detailed in a paper he and collaborators published in the Journal of the American Society for Mass Spectrometry in January.
"I think it's great to see that this is being done," he said. "We know it has been of value for us, and seeing vendors implement it is great."