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Max Planck Team Develops Reagents for Alternative Isobaric Tagging Technique

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NEW YORK (GenomeWeb) – Researchers at the Max Planck Institute of Biochemistry have designed an isobaric tagging reagent that could improve the performance of multiplexed proteomic experiments.

Called EASI-tags (easily abstractable sulfoxide-based isobaric-tag), the reagents are intended for use in a recently developed isobaric tagging technique called complementary reporter ion tagging. That method, developed by Martin Wühr, assistant professor of molecular biology at Princeton University, uses an alternative approach to quantifying tagged proteins that addresses the precursor interference issue that has troubled conventional isobaric tagging workflows.

While Wühr and his colleagues have had some success using the workflow with existing isobaric tagging reagents, its performance would likely be improved by use of reagents optimized specifically for the technique. The EASI-tags, which were described in a paper published this week in Nature Methods, represent one of the first efforts to design such a reagent.

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.

These reagents are sold as tandem mass tags, or TMT, by Thermo Fisher Scientific through a licensing agreement with Proteome Sciences, and as isobaric tags for relative and absolute quantitation, or iTRAQ, by AB Sciex.

Isobaric tagging is commonly used to multiplex proteomic experiments, allowing researchers to analyze several different samples simultaneously, which improves throughput and reduces variation due to differences across LC runs and other factors. By using distinctly patterned isotope tags for each sample, researchers are able to combine them while still being able to keep track of what sample is contributing what signal.

One of the major downsides of isobaric tagging is precursor interference, which can significantly impact the accuracy and precision of quantitative information generated in these experiments. 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 also contribute to the reporter signal for the target peptide, which decreases the accuracy with which the actual target is measured.

To get around this problem, Wühr and his collaborators developed an approach where, instead of measuring the reporter ions cleaved from the labeled peptides, they measure the peptides along with the portion of the reporter ion left attached after fragmentation. This results in reporter ions with different masses for each peptide. And this allows researchers to distinguish between these reporter ions even in the case of co-isolating peptides, which mitigates the interference problem.

Wühr's lab has used TMT reagents, isobaric tags sold by Thermo Fisher Scientific and intended for conventional tagging workflows. However, TMT tags are not well suited to the complementary reporter ion approach.

TMT tags have chemistries that are very similar to that of peptides themselves, said Felix Meissner, a Max Planck professor and senior author on the EASI-tag paper, which was done in collaboration with the lab of Max Planck researcher Matthias Mann.

This means that the TMT tags and the labeled peptides will fragment at similar collision energies, making it difficult to cleave the tag while leaving the peptide intact, and difficult to efficiently form the complementary reporter ions the experiment is meant to measure.

What was needed, Meissner said, was a tag chemistry that could be cleaved at a lower energy level than the peptides. Taking their cue from reagents used in protein crosslinking experiments, he and his colleagues investigated sulfoxide-based reagents, finding that these would fragment at energy levels that left the peptides themselves intact.

"So what you can do is optimize the [collision] energies for two things," he said. "You can have one optimal energy to generate the peptide-coupled reporter species for quantification, and then you can use another [higher] energy to fragment the peptides to get the IDs."

By eliminating the precursor interference issue, the complementary reporter ion approach enables experiments that were not previously feasible with isobaric tagging, Meissner suggested.

"For example, you can have reference channels with one, ten, a thousand proteins, a complete proteome, where you know exactly the amount of each protein present, and then you can use that for absolute quantification, which was not possible before," he said.

Researchers are exploring other approaches to addressing the precursor interference problem, most notably doing quantification at the MS3 level, which adds another level of ion isolation and fragmentation. However, moving to the MS3 level results in a slower duty cycle and the loss of ions, which can lead to a loss of sensitivity, and, Meissner noted, such approaches are best-suited to very high-end mass spec instruments, which currently limits their accessibility.

Wühr, who was not involved in the EASI-tag development, also suggested that the complementary reporter approach needs further optimizing. He said that based on his reading of the Nature Methods study, he suspected that the reagents actually fragmented too easily, leading to highly complex spectra and a reduction in peptide identifications.

He noted that he had not used the EASI-tag reagents himself, but that this had been a problem with a separate similar effort his lab collaborated on with researchers at Munich's Ludwig Maximilian University that likewise explored sulfoxide-based tagging chemistries. 

He said that his comments about the EASI-tags were based on the fact that "the numbers that they show in the [Nature Methods] paper are a little bit underwhelming coming from a fantastic mass spec lab like the Mann lab."

Using the tags to look at a combination of a yeast and a human proteome split into 24 fractions and analyzed using 120 minute LC gradients, the researchers identified a total of 10,009 protein groups — 3,485 from yeast and 6,524 from human.

Wühr said that in similar experiments in his lab using TMT tags, which he noted "are really bad for this kind of approach," his team typically identifies around 9,000 human proteins and 4,000 yeast proteins.

He added that it is possible the lower number identified in the Nature Methods experiment was due not to the fragmentation behavior of the EASI-tags but rather due to the fact that existing mass spec search engines are not optimized for the data produced by such experiments.

Meissner indicated that this was what he believed to be the explanation, noting in an email replying to Wühr's comments that "we would argue that the EASI-tag fragments exactly as we wish," and adding that he and his colleagues were "working on increasing the [identification] rates" through "computational improvements in the data analysis software."

Meissner said his lab is also working to increase the tags' multiplexing capabilities. Currently, they can multiplex up to six samples, compared to a max of 10 samples for conventional TMT labeling experiments.

He said he and his colleagues have patented the reagents and plan to commercialize them. He added that they have received interest from vendors, but have no specific commercialization plans at the moment. He also noted that instructions for synthesizing the tags are included in the manuscript and that labs with organic chemistry expertise should be able to make them.

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