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UW-Madison Team Develops Workflow for 24-Plex Proteomic Experiments


NEW YORK (GenomeWeb) – A team led by researchers at the University of Wisconsin-Madison have developed an isobaric tagging approach that allows multiplexing of up to 24 samples in a single mass spec experiment.

Described in a paper published this month in Analytical Chemistry, the method combines stable isotope dimethyl labeling with DiLeu isobaric tagging to enable high levels of multiplexing. While this level of multiplexing comes at the expense of depth of coverage, the technique could prove useful for experiments where high sample throughput and quantitative data are a priority, said Lingjun Li, professor of pharmaceutical sciences and chemistry at UW-Madison, and senior author on the study.

Called cPILOT (for combined precursor isotopic labeling and isobaric tagging) the technique uses two labeling approaches to increase sample multiplexing. The first, dimethyl labeling, uses the addition of light and heavy formaldehyde and sodium cyanoborohydride to proteins or peptides to create light- and heavy-labelled samples, the basic idea being similar to SILAC labeling, but with the labels being added chemically as opposed to metabolically.

These two sets of dimethyl labeled samples are then isobarically tagged using 12-plex DiLeu tags. When analyzed via mass spec, the two different dimethyl labels can be distinguished between at the MS1 level, while the 12-plex DiLeu tags can be read out at the MS2 or MS3 level, allowing for 24-plex experiments.

As the study authors noted, scientists have done similar studies using TMT isobaric tags, including a pair of studies from Harvard University researcher Steven Gygi that combined SILAC labeling with TMT to multiplex 18 and 54 samples, respectively.

The main advantage of the DiLeu approach versus TMT is price, said Li, adding that for many labs the relatively high cost of TMT reagents presents a barrier to highly multiplexed experiments of the kind presented in the Analytical Chemistry paper, which, Li noted was done in collaboration with the lab of Vanderbilt University researcher Rena Robinson, a co-author.

"By synthesizing the labeling reagents in-house, quantities can be prepared at large scales for experimental designs that would be prohibitively expensive with commercial tags," she said. "An experiment that would cost many thousands of dollars [using TMT] can be performed for under $100 with DiLeu, and that is something many researchers would be very interested in."

Li added that another potential plus of the DiLeu tags compared to TMT is the fact that they more easily dissociate from peptides at lower collision energies, which leads to more intense reporter ions. While not as significant an advantage as the cost savings, she said this "potentially benefits quantitative accuracy and reduces the frequency of spectra with missing channels as multiplexing increases."

The more intense reporter ions could also improve quantitation in isobaric tagging experiments that measure the reporters at the MS3 level to counter the precursor interference problem inherent in isobaric tagging methods.

On the other hand, the readier dissociation of the DiLeu tags could, in some peptides, "generate abundant reporter ions and an abundant tag loss peak in the high mass region, but few backbone fragments," Li said, noting that in such cases these "peptides are not sequenced or are sequenced with low confidence."

Use of the DiLeu tags also involves an extra step to activate the tags given the fact that the active version of the tag will hydrolyze quickly during storage, noted Dustin Frost, a post-doc in Li's lab and first author on the paper.

"We store the DiLeu tags in their stable, inactive carboxylic acid form and activate them immediately prior to labeling to ensure optimal labeling performance, Frost said, noting that this activation reaction takes about 30 minutes followed by an extra strong cation exchange cleanup step to remove activation byproducts and extra DiLeu reagent.

Li and her colleagues developed their first DiLeu tags, which allowed for four-plex experiments, in 2010. However, she said, they are less frequently used outside her lab. She suggested this is due largely to the fact that they are not a commercial product like TMT, and added that the intellectual property covering the TMT reagents "covers isobaric tags in general terms" and prevents other entities from bringing competing isobaric tagging products to market.

TMT was developed by UK-based proteomics firm Proteome Sciences, which has licensed the technology to Thermo Fisher Scientific. In response to a query regarding the IP around TMT and isobaric tagging, Thermo Fisher did not directly address whether commercialization of the DiLeu tags would infringe on the TMT IP but said that "proposals to use the licensed technology requires permission from Proteome Sciences."

Ian Pike, a director at Proteome Sciences, confirmed the company had licensed its TMT patents to Thermo Fisher but did not say whether a commercial version of the DiLeu tags would infringe on those patents.

"It is not our role to determine what constitutes infringement, and I cannot comment on any third-party opinion as to their ability to commercialize their own technology," he said.

Li said that regardless of the IP issues a commercial product might run into, she and other researchers are "free to develop and use DiLeu tags" for their own research. The 12-plex tags used in the Analytical Chemistry study " are synthesized in one or two steps using straightforward chemistries and commercially available starting reagents," she said.

In the study, the researchers tested the 24-plex approach in yeast, analyzing the samples on a Thermo Fisher Fusion Lumos instrument using the SPS-MS3 acquisition approach. Of 1,953 proteins identified, 1,351 could be quantified, and 1,121 were quantified across all 24 samples.

While this is not a particularly deep analysis in terms of proteome coverage, the ability to combine 24 samples in a single experiment makes for high throughput and suggests the approach could prove useful "particularly for large-scale applications in which high sample throughput and quantification may be preferred over deep proteome coverage," the authors noted.

"It's up to the researcher to determine if a hyper-plexed 24-plex analysis — with increased spectral complexity, co-isolation, and redundant sampling for the sake of increased multiplexing — is more appropriate for their experiment than performing multiple separate 12-plex analyses that are free of these things," Li said.

Li also noted that 36-plex analysis was possible with the cPILOT technique. In that case, she said, researchers would include an additional dimethylation label at a mass between the light and heavy labels used in the 24-plex experiment, which would provide light-, medium-, and heavy-labeled samples, each of which could then be tagged with the 12-plex DiLeu reagents.

However, this would further reduce mass spec duty cycle efficiency and proteome coverage. Li noted that her lab was not working on a 36-plex at this time.