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Waters, TUM Researchers Use Ion Mobility to Attack QTOF Duty Cycle Limitations

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NEW YORK (GenomeWeb) – A team led by researchers from Waters and the Technical University of Munich has devised an ion mobility-based method for improving the duty cycle of quadrupole time-of-flight (QTOF) mass spectrometers.

The approach, which was detailed in a study published last month in Molecular & Cellular Proteomics, enables significantly higher sensitivity in QTOF-based bottom up proteomics experiments, providing up to a 10-fold improvement according to Bernhard Kuster, a TUM researcher and senior author on the paper.

In the MCP paper, the authors used the method to analyze HeLa cells on a Waters Synapt G2Si QTOF instrument, identifying 7,500 proteins in a day-long run. Since then, applying the method to a variety of tissue types, the researchers have been able to identify in the range of 5,000 to 8,000 proteins per sample in runs of similar lengths, Kuster told ProteoMonitor.

The researchers also did a phosphoproteomic analysis of A431 skin carcinoma cells, identifying roughly 8,600 phosphorylation sites in five hours of instrument time.

Additionally, they used the method in a chemoproteomic experiment measuring levels of interaction of Trichostatin A with target proteins in K562 myelogenous leukemia cells. The results of this work tracked closely with data generated from the same experiment performed on a Thermo Fisher Scientific Q Exactive instrument, suggesting, the authors noted, that the approach is capable of delivering high-quality quantitative data.

While QTOF instruments are in theory capable of collecting tandem mass spectra at speeds as high as 100 Hz, many of these spectra do not contain enough high-quality information to allow researchers to make good peptide matches, making for an effective speed that is somewhat lower.

This issue, Kuster said, is inherent in the structure of QTOF instruments, which take a continuous beam of ions passed through the upfront quadrupole and inject them into the TOF analyzer using discontinuous pulses. Because of the lower-duty cycle of the injection system shooting ions into the TOF analyzer, many ions are lost during the time between injections.

"You have a continuous beam of ions going through the quadrupole and then you reach where they are injected into the TOF and then it is a discontinuous process – shot, shot, shot, shot," Kuster says. "And between each shot you have to wait, and all of the fragment ions are coming into the TOF at the same time, and while you send some down the [TOF] tube, the others are just lost, and therefore you are losing a lot of the available ions."

This loss of ions, he noted, results in reduced sensitivity, and, consequently, the number of proteins that can be identified in a typical bottom up experiment.

Trapping devices don't suffer from this issue, Kuster said, because they can continue to collect ions until they have enough to generate good spectra. This, he suggested, has been important to the popularity of Thermo Fisher Scientific's Orbitrap analyzer, which, he said, combines reasonable speed with very good sensitivity.

"It's a balance between sensitivity, speed, and how much material you have," he said. "Sensitivity-wise, traps have a natural advantage because you can collect ions for as long as you need to get good spectra."

However, if you have enough material that the loss of some ions isn't so important, "a QTOF can outperform a trap" due to its speed advantage, he added.

Kuster and his colleagues sought to address QTOF's duty cycle challenges by concentrating peptide fragment ions using the Synapt G2Si's ion mobility system.

He and his colleagues first selected precursor peptides using the quadrupole, then fragmented them in the mass spec's collision cell. They then ran these fragment ions through the instrument's ion mobility device, which separates ions based on features like size and charge. This allowed them to package the ions into more or less discrete groups, sorted by their ion mobility characteristics.

"So you are concentrating your ions," Kuster said. "They come out of the ion mobility device in one small compact package, and you can synchronize with the device that pushes ions into the time-of-flight system."

By compacting the ions into small packets concentrated according to their ion mobility characteristics and then synchronizing these packets' injection into the TOF analyzer with their arrival, the researchers were able to lessen the ion loss due to the low duty cycle of the injection system.

As Kuster and his co-authors noted in the MCP paper, other labs have in the past tried similar approaches. But while these efforts have met with some success, they've also had limitations.

For instance, in 2000, researchers at what was then Sciex (now AB Sciex) approached the problem by trapping fragment ions in a QTOF collision cell and releasing them in short bursts synchronized with the TOF injector. However, the MCP authors noted, while this approach offered up to a 100 percent improvement in duty cycle, this level of improvement was achieved over only a small mass range.

The method presented by Kuster and his colleagues appears to work across the full mass range analyzed in typical proteomic experiments, he said, noting that they "have not seen any biases."

While the technique could in theory be performed on other QTOF instruments with ion mobility, Kuster said, it requires that the instrument have an ion mobility device after the collision cell so that the fragment ions, as opposed to the precursors, can be sorted in this manner.

Agilent and AB Sciex, for instance, both offer ion mobility devices with certain of their QTOF instruments, but these devices are typically attached at the front end prior to the collision cell.

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