NEW YORK (GenomeWeb) – Scientists from Bruker and the lab of Max Planck Institute of Biochemistry researcher Matthias Mann has developed a mass spec method using trapped ion mobility spectrometry (TIMS) that could offer significant improvements in speed and sensitivity compared to standard workflows.
Detailed in a paper published last week in the Journal of Proteome Research, the method, named Parallel Accumulation - Serial Fragmentation (PASEF) combines collection of ions via TIMS with rapid quadrupole switching on a QTOF instrument to enable the fragmentation of multiple simultaneously eluting precursor ions.
According to the researchers, the method could enable around a 10-fold increase in mass spec sequencing speed without any loss in sensitivity.
The approach relies on the ability of TIMS to collect ions in parallel and then release them into the mass spec on the basis of their collisional cross section, and the ability of the mass spec's quadrupole to switch rapidly between different masses such that it can isolate a number of different precursors for fragmentation 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 for fragmentation and analysis. Due to limitations of instrument speed, however, the majority of precursors in complex samples are never selected for fragmentation.
Additionally, while increased instrument speed helps up the number of precursors an instrument can analyze in a run, this speed can come at the cost of sensitivity. As the quadrupole switches between different precursors, it is only selecting for any one of these precursors for a short period of time, meaning that, in the case of any particular precursor, the machine is only passing on for analysis a small portion of the total amount of that species present in the sample.
With the PASEF method, on the other hand, 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 precursor 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.
Key to the approach is the ability of the quadrupole to switch at this rapid rate, said Florian Meier, a Max Planck researcher and first author on the study.
"It is actually built on a very simple idea," he told GenomeWeb. "In TIMS you trap the ions and then elute them based on their ion mobility. So now, instead of selecting just one single ion during the elution, we thought if we could switch the quadrupole very rapidly we could catch more than one precursor."
Meier added that because the precursors elute from the TIMS device in compressed packets, the same amount of target ions are being introduced into the mass spectrometer as would be in a normal experiment, despite the rapid switching. This, he noted, means that even with the increase in speed, sensitivity remains the same.
And, in cases where this extra speed is unnecessary for looking at as many different precursors as possible, it can be used to increase sensitivity by looking at the same precursor multiple times in a single scan.
"If you acquire precursors faster and faster, you don't necessarily get increased sensitivity because some of those precursors are too low abundance for detection," Meier noted. "We have this super high sequencing speed, so we can just sequence these precursors several times and then add up these scans and get a higher signal-to-noise ratio than with the single scan — and this we hope will give us even more identifications."
As the authors noted, data-independent mass spec methods have approached the precursor sampling problem from a different perspective by simply fragmenting all the precursors in a sample without first selecting them. Such approaches have proven a great success in recent years, seeing significant uptake by proteomics researchers, but they come with their own issues.
For instance, the multiplexed spectra generated by DIA experiments can reduce the number of peptides identified. Additionally, such methods are not compatible with isobaric labeling techniques like iTRAQ and TMT because the signal from the fragmented isobaric labels can't be linked back to the labeled precursor.
"That is the great thing about this [method]," Meier said. "We do not give up the selectivity of data-dependent methods."
The researchers have not yet, however, been able to actually implement the approach in a large-scale shotgun experiment, noted Meier's Max Planck colleague Scarlet Beck, also an author on the paper.
In a proof-of-principle experiment, they introduced a four-protein digest into a Bruker impact II QTOF attached to a TIMS device, finding that the instrument's quadrupole was able to switch quickly enough to isolate four TIMS-separated precursors. They also observed that the quadrupole windows they used were roughly three-fold longer than they needed to be and that, in fact, at least 10 different precursors could have been selected.
Extrapolating these findings to a large-scale shotgun experiment, the researchers wrote that using PASEF around "70 percent of the overall precursor population can now be targeted and potentially identified by MS/MS."
Before this can be demonstrated in a real experiment, however, modifications to instrument controls and software will need to be made on Bruker's end, Beck said. "We did a proof of principle to show that it is possible, but now we need the implementation from Bruker's side before we can do a shotgun proteomics experiment."
Meier said that he expected this implementation would be accomplished relatively soon.
While the researchers are exploring the technique on the Bruker TIMS and QTOF platforms, it is, in theory, applicable to other ion mobility-QTOF instrument combinations, Beck said — such as systems sold by Sciex and Waters, for instance — so long as those instruments are capable of the rapid quadrupole switching required by the method.