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Princeton Team Develops New Isobaric Tagging Method

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NEW YORK (GenomeWeb) – Princeton University researchers have developed a new isobaric labeling approach for multiplexed proteomic experiments.

Described in a paper published last week in Analytical Chemistry, the method could offer improved sensitivity and accuracy compared to existing methods while also being compatible with a wider range of instrumentation, said Martin Wühr, assistant professor of molecular biology at Princeton and senior author on the study.

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 also 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. Currently, 10 is the maximum level of multiplexing enabled by commercial isobaric tagging reagents.

The approach is not without downsides, though. Most notable is the precursor-interference problem that can lower the accuracy and precision of the quantitative data obtained in isobaric tagging experiments.

In these experiments, the mass spec isolates a 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.

One approach researchers have taken to dealing with this challenge is to do quantification at the MS3 level, which adds another level of ion isolation and fragmentation, reducing the problem of precursor interference. This method has been pioneered in large part by Harvard University researcher Steven Gygi. Proteome Sciences, which manufactures the TMT reagents, has developed three-stage TMTs for use in such workflows.

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. Additionally, MS3 isobaric tagging workflows are best suited to high-end instrumentation like Thermo Fisher Scientific's Fusion Lumos mass spec, which limits their accessibility currently, said Wühr, who, as a postdoc in Gygi's lab was co-author on a 2014 Analytical Chemistry paper that presented an MS3 isobaric tagging method.

Given these challenges around MS3 isobaric tagging workflows, Wühr and his colleagues tried a different approach to the problem. Instead of measuring the reporter ions produced by the fragmentation of the isobaric tags in MS2, they measured the peptides plus the portion of the reporter tag still left attached to that peptide after fragmentation. In this way they were able to retain the distinct isobaric tag information that allowed multiplexing of different samples, while also measuring the peptide it was attached to. This results in reporter ions, which they termed TMTc 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 essentially eliminates the interference problem.

The idea for this approach actually stemmed from his initial misunderstanding of isobaric tagging, Wühr said. "To be perfectly honest, when [Gygi] first explained to me how TMT is supposed to work, I misunderstood it," he said. "I thought we were not looking at the part [of the reporter ion] that falls off but at what we now call the [TMTc] ions."

In 2012, Wühr, Gygi, and colleagues published a paper in Analytical Chemistry presenting the TMTc method. This month's Analytical Chemistry study presents an updated version of the approach called TMTc+ that addresses several problems with the initial method.

One major problem with the initial method was precision due to the deconvolution process involved in extracting the different peptide measurements made in each of the different samples being multiplexed. To address this issue, the researchers developed a method wherein they measured the isolation window being used for a given experiment and incorporated this measurement into their deconvolution algorithm, an approach that Wühr said has pushed the precision of the TMTc+ approach nearly in line with that achieved by standard MS2 or MS3 isobaric tagging experiments.

The other issue was that for many peptides, TMTc ions did not form efficiently, which significantly limited the method's sensitivity. This was particularly a problem for larger peptides with higher charge states, Wühr said. The TMTc+ approach tackles this problem by switching from a LysC-only protein digestion to digestion using trypsin and LysC, which produces smaller peptides on average. The researchers also added DMSO, which moves the ion charge states toward 2+.

With these improvements, the TMTc+ method appears to outperform both the MS2 and MS3 methods in terms of protein quantitation, Wühr said. In an analysis looking at unfractionated HeLa lysate using a three-hour LC gradient, the researchers quantified 3,882 proteins using TMT-MS2, 3,729 using TMTc+, and 2,739 using TMT-MS3, with the slightly higher number of proteins quantified via the MS2 method offset by the precursor interference issue.

"I would argue that this is the best possible data you can get currently for multiplexed proteomics," Wühr said, adding that his lab now primarily uses the TMTc+ approach for its isobaric tagging work.

One remaining drawback of the TMTc+ approach is its limited multiplexing, the authors noted. While MS3 TMT workflows can combine up to 10 samples in an experiment, the TMTc+ method is limited to five samples.

The authors wrote, however, that 10-plex experiments could be plausible with new tagging reagents developed specifically for the TMTc+ approach.

Wühr added that new reagents tailored to the TMTc+ method could further improve the method's sensitivity.

"The TMT tags were really not made for this purpose," he said. "They don't have the right bond strengths for it. It's kind of an accident that these complement ions form at all."

He declined to say if either Thermo Fisher or Proteome Sciences are working on TMT reagents intended for TMTc+ assays but noted a study published last week by his lab and collaborators at Munich's Ludwig Maximilian University in which they presented an isobaric tag developed for the TMTc+ method.

That reagent, "is not yet ready for prime time," Wühr said, "but we are continuing to work closely with the [LMU] group to make a tag that is really well suited for this, and we will then be able to get even better data."

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