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Mass-Spec Vendors Explore Use of FAIMS as Additional Separation Technique for LC-MS Workflows

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By Adam Bonislawski

With proteomics researchers continuing to seek more efficient ways to reduce sample complexity prior to mass spec analysis, a number of scientists and instrument companies are exploring the use of field asymmetric ion mobility spectrometry, or FAIMS, as a separation technique.

Large vendors including Thermo Fisher, Agilent Technologies, AB Sciex, and Bruker are either developing FAIMS devices for use with mass spec or have recently presented research on use of the technology, which has the potential to add significant separating power to conventional LC-MS workflows.

FAIMS works by running mixtures of ions at atmospheric pressure between two electrodes, 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.

"By filtering out abundant signals using a FAIMS device, you allow yourself to accumulate lower-abundance signals," Mike MacCoss, assistant professor at the University of Washington's Department of Genome Sciences, told ProteoMonitor. "You have the LC separation, and then you have the FAIMS separation, and then you have the mass spec separation of each of those FAIMS. So it just adds an extra dimension onto the separation."

MacCoss is currently collaborating with Thermo Fisher, which has commercially available FAIMS devices, to develop applications for analyzing peptides using FAIMS as part of an LC-MS platform, and has been investigating the use of FAIMS with the company's ion trap instruments. The primary advantage to adding the technology to LC-MS workflows, he said, is the time it saves on separations and mass spec analysis.

"Biochemical fractional [distillation] could work as well, but it's done on a slower time scale and often results in more mass spectrometry time because you have to run many different LC fractions," he said. "Say you took 12 strong cation-exchange [LC] fractions and ran them on 12 reverse-phase [LC] gradients. It takes two hours to run your [LC] gradient – that's 24 hours. With FAIMS we can do that in the same time as one LC run."

There's reason to believe that this is just the start of FAIMS' separating capability, as well. While devices like Thermo Fisher's have been on the market for several years, they aren't able to scan ions quickly enough to keep up with the pace of typical LC systems. Using them in LC-MS workflows requires stepping in large increments, which shortens the FAIMS cycle but also lowers the resolution, reducing the gain in separation the technique provides.

To take full advantage of FAIMS' separating capabilities, researchers at the Pacific Northwest National Laboratory and Cambridge, UK-based biotech firm Owlstone Nanotech have been working to miniaturize the devices, which allows for the use of more intense electric fields, which, in turn, increases separation speeds.

"The efficiency of FAIMS scales as roughly the cube of the field, so if you double the field, you accelerate the speed [of the FAIMS device] 10 times," Alexandre Shvartsburg, bio separations and mass spectrometry scientist at PNNL, told ProteoMonitor. "So that is potentially a solution to the problems in using FAIMS in proteomics and other biological analyses."

"There has been an inherent mismatch in the timescale between LC and FAIMS," he said. "[Researchers] needed to either to dumb down the FAIMS – step on the FAIMS three or four steps rather than capture its full separation power – or slow down the LC, which means your throughput slows way down."

By bringing FAIMS down to the microchip level, Shvartsburg said, PNNL and Owlstone researchers have been able to dramatically increase the intensity of the electric field used and create a device capable of separating LC elutions without stepping.

"That's allowed us to do LC-FAIMS-MS on a time scale that is the same as regular LC, so there's no slowdown and whatever separation power FAIMS provides can be fully captured," he said. "In fact, [the device] is even too fast. It can loop multiple times over the elution of an LC peak and still stay within that peak. It's not a limiting point anymore."

According to Owlstone project manager Danielle Toutoungi, the company has developed a prototype of the microchip FAIMS device in partnership with Agilent. The two companies announced a collaboration to explore FAIMS/TOF-MS applications last June.

Several of the prototypes have been sent to academic groups and biotech firms for testing, Toutoungi told ProteoMonitor. Researchers from Loughborough University will be publishing work in an upcoming edition of Analytical Chemistry investigating use of the device with a Thermo Fisher LTQ ion trap and an Agilent 6230 TOF instrument to separate singly and multiply charged peptides prior to mass spec analysis.

The Owlstone prototype, Toutoungi estimated, is able to scan ions at roughly 10,000 times the speed of FAIMS devices now on the market. The next version of the device, which the company is currently working on, should be even faster, she noted.

"The way people tend to use [currently available] devices is by doing some pre-experiments where they work out what voltage to use to get ions of interest through and then have maybe two or three that they've picked and just cycle through those two or three settings," she said. "That means you're just kind of losing everything else that would come through between those values. We can do complete sweeps, which means you don't lose stuff. Everything comes through; it just comes through at different times."

While Owlstone's FAIMS device was developed in collaboration with Agilent, Toutoungi said it could be produced for other vendors' machines, as well.

"It should actually be a very simple instrument to put onto your source," she said. "Once it's productized it will be very user friendly. It doesn't take a lot of optimization up front to work out what settings to use."

Whether the company will seek to sell the device exclusively through mass spec vendors or as an independent product is still "up in the air," Toutoungi said. "I don't think we'd rule out either at this point."

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