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Thermo Fisher Gives FAIMS Second Try With New Release

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NEW YORK (GenomeWeb) – Thermo Fisher Scientific has launched a new High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS), bringing ion mobility capabilities to the company's mass spectrometers.

Announced last month at the 2018 International Mass Spectrometry Conference in Florence, Italy, the release is a reboot of sorts for the company, which launched a FAIMS device more than a decade ago but saw little uptake among researchers.

FAIMS works by running mixtures of ions at atmospheric pressure between two electrodes and subjecting them to alternating high- and low-field conditions by applying an asymmetric RF waveform, which separates them based on differences in ion mobility under high and low electric fields. Depending on their mass and charge, some ions will drift into the electrodes while others will pass through the device, allowing, for instance, a researcher to select low-abundance peptides for analysis from a tryptic digest while weeding out higher-abundance ions.

The technology can be inserted between a traditional liquid chromatography system and a mass spec instrument, allowing for an additional level of separation.

Thermo Fisher's original FAIMS device had several drawbacks that prevented it from becoming a commercial success, said Iain Mylchreest, the company's vice president, R&D, analytical instruments. Among the most notable was poor transmission of ions from the FAIMS device into the mass spec.

"We could lose almost an order magnitude in signal with the [original] device attached to the [mass spec] system," Mylchreest said, noting that this presented obvious challenges for the sort of high-sensitivity proteomics workflows the technology was meant to enable.

Additionally, he said, the device's cycle time was on the order of hundreds of milliseconds, while the mass spec's cycle time was on the order of tens of milliseconds, meaning the FAIMS device was not able to supply ions fast enough to take full advantage of the mass spec's speed.

The original device was also poorly compatible with the nanoflow and microflow LC systems commonly used by proteomics researchers, Mylchreest said.

The new device, called the FAIMS Pro interface, addresses these issues, he said, noting that it now manages an overall ion transmission rate typically great than 50 percent and in many cases greater than 80 percent, depending on the experimental conditions used. In 2014, Thermo Fisher researchers published a paper in the Journal of the American Society for Mass Spectrometry detailing a new ESI-FAIMS interface that offered a ninefold improvement in ion transmission and provided for an overall ion transmission rate of around 70 percent.

The company has also brought the cycle time down to the tens of milliseconds range and made it compatible with nano- and microflow LC devices, Mylchreest said.

These improvements have been detailed in several recent studies by Thermo Fisher scientists and outside researchers using the new device. In July, researchers at the University of Wisconsin-Madison who published a paper in Analytical Chemistry used the device with an Orbitrap Fusion Lumos mass spec to analyze the proteome of the human cell line K562. In a five-hour experiment using no fractionation, the researchers were able to identify more than 100,000 peptides and more than 8,000 proteins.

The researchers also looked at whether the FAIMS Pro could effectively substitute for sample fractionation, finding that a six-hour, single-shot FAIMS experiment identified 8,007 proteins compared to 7,776 proteins for an experiment that ran a sample separated into four fractions using four 90-minute gradients. The result suggests that FAIMS could replace modest amounts fractionation, said UW-Madison researcher Joshua Coon, senior author on the study.

Also in July, a team led by researchers from the University of Montreal published a study in Molecular & Cellular Proteomics using the FAIMS device coupled to a Thermo Fisher Orbitrap Fusion Tribrid, finding that it boosted peptide identifications by 30 percent while extending the limit of detection by nearly an order of magnitude in an analysis of HEK293 cell digests.

The researchers also found that use of the device reduced the amount of precursor interference, which, as they noted, is helpful in proteomic experiments generally and particularly helpful in quantitative experiments using isobaric tagging where precursor interference is a significant challenge.

In an isobaric tagging experiment, the mass spec isolates the target ion and fragments it, generating the isobaric tag reporter ions that correspond to the proportions of the different peptides in the tagged samples. However, the isolation windows used to target a given precursor ion are typically wide enough that other non-target ions can slip through. Because these ions have also been labeled with isobaric tags, they contribute to the reporter signal for the target peptide, which decreases the accuracy with which the actual target is measured.

Researchers have adopted a variety of methods to address this problem, one of the most effective of which has been to do an additional round of fragmentation and measure the ions at the MS3 level, which should eliminate much of the interference issue. This approach suffers, however, from a longer cycle time, which reduces the number of peptides the instrument can quantify.

In their study, the University of Montreal team, led by Pierre Thibault, professor of chemistry and senior author on the MCP paper, found that the use of FAIMS to reduce precursor interference in a 10-plex TMT analysis of HEK293 cells boosted the number of proteins quantified by 2.5-fold compared to an MS3-based approach.

This followed a 2016 paper published by Thibault and his colleagues in the Journal of Proteome Research in which they found a version of the new FAIMS technology increased the number of peptides they could quantify in a TMT analysis of HEK293 cells by 68 percent compared to a standard MS2-level TMT analysis while also improving the accuracy of their quantitation.

Mylchreest said he envisioned several main areas where the technology could prove useful. The first are large-scale qualitative and quantitative proteomic analyses of the kind done by the Coon and Thibault teams where FAIMS could improve depth of coverage and accuracy of quantitation.

Another area where the device could see uptake is for targeted quantitation assays where it could help reduce interferences hampering protein or small molecule measurements, he said. It could also prove useful for top-down and intact protein work, he further noted.

With release of the FAIMS Pro, Thermo Fisher joins the majority of other major mass spec vendors that offer ion mobility technology with their instruments. Waters, Sciex, Agilent, and Bruker each offer some version of ion mobility with various versions of their mass specs.

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