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FAIMS Gaining Ground as Proteomics Separations Tool as Vendors, Researchers Push Development

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This story has been corrected to reflect the fact that Waters, not Thermo Fisher, was the first large mass spec vendor to incorporate a FAIMS device.

After years spent lingering outside the mass spec mainstream, field asymmetric ion mobility spectrometry, or FAIMS, is growing increasingly popular as a separations technique for a variety of applications, including proteomics.

The technology remains a niche player in proteomics research (PM 10/29/2010), but recent developments – including AB Sciex's release in 2011 of its new FAIMS-based Selexion technology and first-ever sessions dedicated to the technique at this year's Pittcon and American Society for Mass Spectrometry annual meetings — suggest that its reach is growing.

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.

Waters was the first large mass spec vendor to incorporate a FAIMS device, licensing the technology in 2003 from biotech firm Ionalytics. After Waters chose not to renew the license, Thermo Fisher Scientific -- then Thermo Electron -- acquired Ionalytics and released a FAIMS device in 2006. That device suffered, however, from problems with reproducibility, speed, and resolution, said Alexandre Shvartsburg, bio separations and mass spectrometry scientist at the Pacific Northwest National Laboratory and a leading FAIMS researcher.

"It was a little early and there were some well-known issues with implementation," he told ProteoMonitor, "and [Thermo Fisher] never got very far with it."

Last year, AB Sciex became the latest major vendor to introduce a FAIMS-based separations technique with the release of its Selexion technology, which it has incorporated into its Triple Quad 5500 and 6500 and QTRAP 5500 and 6500 systems.

This version of FAIMS, Shvartsburg said, offers improvements over the Thermo Fisher device in terms of reproducibility, speed, and resolution, generating renewed and new interest in the technology.

The Selexion release "has been maybe the most important and consequential" development in terms of driving interest in FAIMS, he said, noting that at this year's ASMS AB Sciex and its collaborators showcased dozens of presentations on research using the technique.

"[Selexion] is a very powerful product, and it's easy to see why it took off so well," he said.

Agilent and Bruker are also working on incorporating FAIMS devices into their mass specs, Shvartsburg noted – the former with UK-based Owlstone Nanotechnology and the latter with collaborators including University of North Carolina researcher Gary Glish.

Agilent and Owlstone announced a collaboration in June 2009 to explore FAIMS/TOF-MS applications. The Owlstone device, which was in part developed in collaboration with Shvartsburg and his colleagues at PNNL, is designed to fit into an Agilent Jet Stream source, although, said Owlstone project manager Danielle Toutoungi, it can work upfront of other vendor's machines as well.

Toutoungi said that Agilent hasn't formally announced any plans to release instruments incorporating the technology, but, she noted, the company presented on it at this year's ASMS, "which usually indicates that things are happening."

According to Shvartsburg, the key advantage of the Agilent-Owlstone device is its speed, which he called "just amazing." High speed enables the FAIMS system to operate at full separating power while still keeping up with standard-flow LC timescales – something, he said, the Thermo Fisher and AB Sciex devices aren't able to do.

The Sciex instrument, on the other hand, offers superior resolution compared to the Thermo Fisher and Agilent-Owlstone devices, while the Thermo Fisher device still bests both of them in terms of sensitivity due to the fact that its cylindrical design transfers ions into the mass spec better than do the other vendors' planar FAIMS systems.

"At this point there are basically three platforms [released or nearing release]," Shvartsburg said. "If you draw a triangle with three analytical measurements – resolution, sensitivity, and speed – on the speed side the Owlstone product is the product of choice; on the resolution side the AB Sciex product would be your choice; and on the sensitivity side there is still nothing that beats the [Thermo Fisher] product."

He added that he and his team also continue to improve their own in-house FAIMS systems, focusing primarily on adding resolution. The PNNL team recently submitted a paper to the Journal of Physical Chemistry demonstrating a system with resolving power of 500, a level higher than "any commercial system," he said.

To date, both AB Sciex and Agilent have presented most extensively on the use of FAIMS for small molecule analysis, but Shvartsburg said, the technology is gaining traction in proteomics, as well.

According to Robert Moritz, a researcher at the Institute for Systems Biology, one reason for slow adoption of FAIMS by proteomics researchers had been the difficulty of interfacing the device with nanoscale ESI systems.

His group, he noted, recently published a paper in Molecular & Cellular Proteomics in which they interfaced a Thermo Fisher Scientific FAIMS unit with a nanoscale ESI system.

"Anyone in the proteomics field would [traditionally] just walk away from [FAIMS] straight away when they saw it didn't do low flow rates," Moritz told ProteoMonitor. "But we've gotten it to work [with nano-ESI] very successfully, and we've tried to show everyone else how to do that as well by releasing a paper with all the diagrams of how to do that."

Using the system, Moritz and colleagues including Kristian Swearingen achieved a more than 50 percent increase in proteome coverage, he said. "You lose a little bit of signal with FAIMS, but you gain so much by increasing the separation between the background noise and the peptides of interest."

Shvartsburg said that analysis of protein post-translational modifications was an area where the technology had recently shown significant potential. He cited in particular a study on using FAIMS to look at histone modifications that he and Northwestern University researcher Neil Kelleher published last month in Analytical Chemistry.

In the study, the researchers used FAIMS combined with mass spec to distinguish between different patterns of acetylation on histone tails. The technique, Shvartsburg said, proved quite effective for this purpose, offering a level of resolution impossible to obtain via LC alone.

Inspired by the success of this work, the researchers then turned to histone methylation and found that – despite this modification's smaller size – they were able to easily resolve different methylation patterns, as well. That work, Shvartsburg said, is currently in review at Analytical Chemistry.

He also noted a recent paper in Analytical Chemistry by University of Birmingham researcher Helen Cooper using FAIMS to look at protein glycosylation.

He suggested that glycosylation analysis could potentially be a "killer application" for FAIMS, "given that localizing glycosylations by tandem mass spectrometry and LC is exceedingly difficult because of how labile they are."

How widely the technology is ultimately adopted still remains to be seen, of course.

"There does seem to be growing interest in [FAIMS], Toutoungi told ProteoMonitor. But, she added, "I think people are waiting to see what can be done with it."

Moritz suggested that weak promotional efforts on the part of vendors have historically played a role in the technology going overlooked. "Companies like Thermo Fisher just don't promote the technology, and most users just don't know it exists," he said.

Ian Jardine, Thermo Fisher's chief technology officer, somewhat conceded Moritz's point, telling ProteoMonitor that while the company "certainly hasn't dropped [FAIMS] by any means … in terms of all the other things we're developing, I wouldn't say it's a first-line priority."

He noted that problems with sensitivity and reproducibility have limited FAIMS' adoption in the past. Another key issue is that it is very difficult to predict, without experimental evidence, how a molecule will behave when submitted to FAIMS separation.

"At this time we don't have predictive capabilities to predict what the separation properties will be for different things, Shvartsburg said. "So some things we think should separate do not separate and other things we think could not possibly be separated separate extremely well."

"I think we've seen [FAIMS] as being more of an esoteric tool," Jardine said. "If your problem is important enough — say quantitation of a particular molecule and you have some interference issues — then you'll spend the time and maybe you'll get a big plus out of this technology. But quite frankly most people want kind of generic techniques that they can just turn on when they need them."

"There seems to be more interest [currently] in [FAIMS]," he said. "But it also seems to come and go. I would say that we have been watching this very carefully."

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