NEW YORK (GenomeWeb) – Researchers at the University of Montreal have developed a mass spec workflow that uses high-field asymmetric waveform spectrometry (FAIMS) to improve quantitative protein measurements in isobaric labeling experiments.
The method, described in a paper published this month in the Journal of Proteome Research, uses the increased separation provided by FAIMS upfront of mass spec analysis to reduce the precursor interference problems associated with the use of isobaric labeling in quantitative proteomics experiments, Pierre Thibault, a University of Montreal researcher and senior author on the paper, told GenomeWeb.
Thibault also suggested that the study demonstrates FAIMS is now a reliable and straightforward enough technology to be incorporated as part of standard proteomics workflows. He and his colleagues used a FAIMS device produced by Thermo Fisher Scientific, which has seen relatively limited uptake among proteomics researchers due to issues with its performance and implementation.
Thibault said this week that while these challenges limited demand for the Thermo Fisher device, the company's current FAIMS system "is easier to operate… and I am sure you will now see a higher number of users who will be interested in the benefit that FAIMS can provide." He noted that he and his colleagues use the Thermo Fisher FAIMS system coupled to the company's Orbitrap Elite mass spec regularly for their proteomics work.
A form of ion mobility, 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 — called Thermo Electron at the time — acquired Ionalytics and released a FAIMS device in 2006, but, as noted, the system failed to catch on with proteomics researchers.
More recently, Sciex introduced a FAIMS-based system with the 2011 launch of its Selexion technology, which it has incorporated into various of its mass spectrometers.
In the Journal of Proteome Research study, Thibault and his colleagues used FAIMS to address the problem of precursor interference when using isobaric labels like TMT reagents for quantitative proteomics experiments.
Isobaric labeling uses stable isotope tags attached to peptides of interest to enable relative or absolute quantitation of proteins via tandem mass spectrometry. Digested peptides are labeled with tags that fragment during MS2 to produce signals corresponding to the amount of peptide present in a sample.
However, other precursors can fall into the fragmentation window for a given peptide, and the reporters released by these additional precursors can interfere with the reporters released by the target peptide, making quantitation unreliable in some cases.
Increasing instrument speed has allowed researchers to address this issue by performing another round of fragmentation and then measuring the tags at the MS3 level. That additional level of fragmentation requires additional analysis time, which can limit sensitivity and proteome coverage.
With that in mind, the University of Montreal researchers investigated if using FAIMS to provide an additional layer of separation could ease the precursor interference challenge. In an analysis of TMT-labeled yeast and human protein digests, they found that FAIMS significantly reduced interfering ions, increasing the number of peptides they could quantify by 68 percent while also improving the accuracy of their quantitation.
Thibault noted that he and his colleagues didn't compare their FAIMS-based approach directly to the MS3 methods, but said that, in theory, making measurements at the MS2 as opposed to MS3 level should allow researchers to acquire more spectra and quantify more peptides. He added that FAIMS could be included as part of MS3 workflows if even more separation is required.
In addition to the TMT work, Thibault and his team have also published on use of the device for phosphoproteomics research where, he said, FAIMS can improve analysis by, for instance, separating phosphoisomers, where phosphate groups are present in different locations on the same peptide.
The technique can also be useful for more narrowly targeted analyses, Thibault said, for instance, in studies that aim to look at small numbers of biomarkers with high sensitivity.
"You can tune in to the appropriate [FAIMS setting] and only look at specific ions, and this obviously significantly improves sensitivity," he said.
A number of mass spec vendors offer ion mobility systems, and firms like Waters, Agilent, and Sciex have heavily marketed their ion mobility capabilities, promoting them as key aspects of their mass spec platforms and developing workflows both in house and through external collaborations that leverage these systems.
Thermo Fisher, on the other hand, has done less visible promotion of its FAIMS systems. In previous interviews, researchers have suggested this is due in part to the technology's limitations. For instance, noted Alexandre Shvartsburg, bio separations and mass spectrometry scientist at the Pacific Northwest National Laboratory and a leading FAIMS researcher, initial versions of Thermo Fisher's FAIMS device suffered from problems with reproducibility, speed, and resolution.
"It was a little early and there were some well-known issues with implementation," he told GenomeWeb in a 2012 interview.
Thibault suggested, though, that it was perhaps time for a reappraisal of the company's device.
"The technology hasn't been pushed by Thermo, but I am sure that in the future it will be something that is of significant [researcher] interest," he said.