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FAIMS Carving Out Niche in Label-Based Proteomic Quantitation Among Early Users


NEW YORK (GenomeWeb) - Researchers at the University of Montreal have used Thermo Fisher Scientific's recently released High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) device to improve the performance of SILAC-based quantitative proteomics experiments.

Described in a paper published last week in the Journal of Proteome Research, the study also demonstrated that the company's FAIMS device, called the FAIMS Pro, could offer depths of coverage equivalent to workflows using moderate amounts of fractionation, which typically adds time and complexity to a proteomic experiment.

The study, along with other recently published work from the lab of Harvard University professor Steven Gygi, indicates that among early users the technology is carving out a niche with label-based quantitative proteomics experiments, helping reduce sample complexity and interferences that can affect the accuracy of these experiments.

In the case of SILAC labeling, which allows researchers to run two or three samples simultaneously and compare protein expression levels across them, co-eluting isobaric peptides can reduce quantitative accuracy. Additionally, like proteomic experiments generally, high-abundance peptides can take up much of the instrument scan time, limiting the identification and quantification of lower abundance peptides.

Traditionally, researchers have used fractionation, in which samples are split into fractions and run separately, to reduce complexity and interferences. However, this can significantly lower throughput and adds sample handling steps that can impact reproducibility.

Ion mobility (IMS) offers researchers an additional layer of separation. Typically inserted between liquid chromatography and the mass spec, IMS separates ions based on their behavior in the gas phase. FAIMS specifically 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.

Companies like Waters, Agilent, and Sciex have for years offered various IMS technologies with their mass specs (Bruker began offering IMS more recently with the launch of its TimsTOF Pro). Thermo Fisher's launch last year of the FAIMS Pro marks its second attempt to market a FAIMS device after an effort roughly a decade ago failed due to a variety of technical issues.

Pierre Thibault, professor of chemistry at the University of Montreal and senior author on the JPR study, said that his lab is currently using the FAIMS Pro "in all cases where we want to improve our quantitative capability."

In the JPR study, the researchers used SILAC to analyze HEK293 cells both with and without FAIMS, identifying 10,752 unique peptides and quantifying 10,146 with FAIMS and identifying and quantifying, respectively, 3,956 and 3,241 peptides without FAIMS. Using upfront fractionation (samples split into five fractions) without FAIMS, they identified and quantified 8,530 and 7,688 peptides, respectively.

The results indicate that FAIMS can effectively substitute for moderate levels of fractionation, which is similar to findings from a study published in July by researchers at the University of Wisconsin-Madison, though the Montreal team found their FAIMS analysis actually improved upon the fractionation-based workflow, while the UW-Madison researchers found that the two were essentially equivalent.

Thibault said his team is also using the device for much of its isobaric labeling work, noting that it is particularly useful for highly multiplexed tandem mass tag (TMT) experiments.

TMT reagents currently allow for multiplexing of up to 10 samples in a single experiment, but this can come at the expense of depth of coverage and quantitative accuracy.

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 when the actual target is measured.

By adding an additional level of separation, FAIMS reduces these interferences. In previous work, Thibault and his colleagues found that FAIMS boosted the number of peptides they could quantify in a TMT analysis of HEK293 cells by 68 percent compared to a standard TMT analysis while also improving the accuracy of their quantitation.

Recent work by Harvard's Gygi lab suggests that FAIMS could also improve the MS3-based TMT workflows developed by his lab. Adding an extra level of fragmentation and measuring the ions at the MS3 level helps alleviate isobaric labeling's precursor interference problems, but the approach requires a longer cycle time, which reduces the number of peptides the mass spec can quantify.

In a study published in Analytical Chemistry in January, Gygi and his colleagues found that both MS2 and MS3 TMT workflows decreased precursor interference by up to sixfold without reducing depth of coverage. The researchers also determined optimal settings of the FAIMS Pro device for different kinds of isobaric labeling workflows.

While SILAC and TMT labeling are among the most popular techniques for label-based quantitative proteomics experiments, uptake of label-free data independent acquisition (DIA) mass spec techniques for quantitative proteomics is growing rapidly.

Researchers including Coons have suggested the possibility of using FAIMS as part of DIA-based quantitative experiments, but little has been published on such an approach thus far. Because DIA experiments typically fragment large swaths of a sample's m/z range at the same time, they also face challenges presented by co-eluting peptides that produce complicated fragmentation spectra that can limit depth of coverage and quantitation.

Thibault said that his lab has not yet tested FAIMS with DIA, but the two techniques should be "totally compatible" and could potentially improve results. He noted, though, that one challenge would be aligning the duty cycle of the FAIMS device with that of the mass spec.