NEW YORK – A team led by researchers at the Max Planck Institute of Biochemistry has developed a mass spec workflow for Bruker's timsTOF Pro instrument that combines the parallel accumulation–serial fragmentation (PASEF) method with data-independent acquisition (DIA).
Detailed in a paper published this week in Nature Methods, the method, called diaPASEF, allows for more comprehensive sampling of peptide precursor ions, resulting in more sensitive analyses.
Scientists in the lab of Max Planck researcher Matthias Mann, senior author on the publication, developed the PASEF approach five years ago. The method takes advantage of the timsTOF Pro's trapping ion mobility spectrometry (TIMS) system, which allows the instrument to collect ions in parallel and then release them into the mass spec analyzer based on their collisional cross section. Combining this with the ability of the instrument's quadrupole to rapidly switch between different masses allows researchers to isolate a number of different precursor ions for fragmentation and analysis in a single scan.
In a typical QTOF-based shotgun mass spec experiment, multiple precursors come off the LC column at the same time and a portion of these precursors are selected by the quadrupole. Due to limitations of instrument speed, however, the majority of precursors in complex samples are never selected for fragmentation and so go unanalyzed.
Increased instrument speed helps boost the number of precursors an instrument can analyze in a run, but this speed can come at the cost of sensitivity. As the quadrupole switches between different precursors, it only selects for any one of them for a short period of time, meaning that only a small portion of that species enters the machine for analysis.
With the PASEF method, precursor ions accumulate in the TIMS device and are then released in discrete packets based on their collisional cross sections. Then, instead of looking at only one set of precursors per scan, the instrument's quadrupole is set to rapidly switch from one precursor to another, allowing the mass spec to analyze multiple precursors per scan, which increases the speed of MS/MS analysis by the number of precursors multiplexed.
PASEF was originally implemented as a data-dependent acquisition mass spec workflow, but the approach was a natural fit for DIA-style analyses, as well, said Ben Collins, a researcher at the School of Biological Sciences at Queen's University in Belfast and an author on the Nature Methods study.
Prior to moving to Queen's University in 2019, Collins was a researcher at ETH Zurich where he had done his postdoc in the lab of Ruedi Aebersold, a leading figure in the development of DIA methods and also an author on the Nature Methods paper. Collins said that he and his colleagues at ETH Zurich had been following the Mann lab's work on the PASEF technique, thinking that it could work well as part of a DIA workflow. Upon learning that the Mann lab was also exploring this idea, the researchers struck up a collaboration, with the ETH Zurich team bringing its DIA experience to the table.
In DIA mass spec, the instrument uses its quadrupole to cycle through broad m/z windows, fragmenting all the precursors in each window. This has proven more reproducible than DDA, where precursors are selected stochastically for fragmentation. However, while the quadrupole is focused on one m/z window, ions outside that window are not selected, meaning the mass spec is only able to sample a small portion of the ions being produced in any given experiment. In the Nature Methods paper, Collins and his co-authors put the ion sampling efficiency of a typical DIA experiment at around 1 percent to 3 percent.
Key to the diaPASEF approach is the fact that an ion's collisional cross section corresponds roughly with its m/z. This means that researchers can accumulate precursors in the TIMS device and then release the ions with m/z matching the DIA window being fragmented at a given moment, while retaining the ions that don't fit within that m/z window. In this way, the method is able to make use of almost all the peptide precursor ions, upping the assay's sensitivity.
Collins noted that in addition to making more efficient use of the ions, the ion mobility provides an extra level of separation that removes interferences and improves signal to noise. The collision cross section [CCS] data generated by the ion mobility separation can also be used as another dimension of information for making peptide identifications.
"We store this [CCS] value in our library along with all the other stuff you would have in a DIA analysis library, like m/z and the relative fragment ion intensities and [LC] retention time," Collins said.
Using the technique to analyze HeLa cell digests, the researchers were able to quantify an average of 4,813 proteins per run using 200 ng of sample and a 21-minute LC gradient, which allowed them to run 60 samples per day. Of those proteins, 4,255 were quantified across those runs, with a coefficient of variation of less than 20 percent.
Reducing LC separation time to increase throughput to 200 samples per day, they were able to quantify more than 3,000 proteins with a median coefficient of variation of 10.3 percent.
The technique also allowed for quantification of large numbers of proteins in very small samples. Running 10 ng of HeLa digest on a 120-minute LC gradient, Collins and his colleagues quantified 4,310 proteins with median coefficients of variation of 9 percent.
Collins said that his lab uses the approach as one of its main mass spec workflows but added that there is still room for improvement.
"I think there are optimizations to the methods that could be imagined in terms of how the quadrupole is synchronized with the ion mobility, in terms of the size of the [fragmentation] windows and the kind of pattern you apply there," he said, noting that the technique could probably benefit from smaller precursor isolation windows or variable windows that narrow as the instrument cycles through more precursor-dense m/z ranges.
He added that further examination of how different peptides, including modified peptides, behave in the TIMS device could also be interesting. He cited recent work by Albert Heck, head of the biomolecular mass spectrometry and proteomics group at Utrecht University, that found TIMS could help isolate cross-linked peptides as an example of the potential of this approach.
"If you have a timsTOF instrument, I think this is going to be the way to go, and we actually see some labs picking it up already," Collins said.