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UW-Madison Team Uses NeuCode Labeling for 18-Plex Mass Spec Experiment

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NEW YORK (GenomeWeb) – The lab of University of Wisconsin-Madison researcher Josh Coon has published a study demonstrating the use of its NeuCode protein labeling technology to multiplex 18 samples in a single mass spec experiment.

Described in a paper published last week in Molecular & Cellular Proteomics, the study combined the NeuCode approach with dimethyl labeling to characterize the salt stress response of five yeast mutant strains at three different time points.

The researchers also compared the performance of the NeuCode technique to isobaric labeling approaches, finding, Coon told ProteoMonitor, that NeuCode delivered similar numbers of identified and quantified proteins but better accuracy and dynamic range.

Coon and his team introduced the NeuCode approach last year in a paper published in Nature Methods. The technique uses differences in the nuclear binding energy – the energy needed to break a nucleus up into its component nucleons – of different isotopes to label amino acids.

Because every isotope has a unique nuclear binding energy, these differences can be used to distinguish between them. NeuCode takes advantage of this phenomenon by incorporating distinct isotope combinations into lysine molecules, which can then be used to metabolically label proteomics samples.

Using different isotopes of carbon, hydrogen, nitrogen, and oxygen, the researchers worked out 39 distinct isotopologues for lysine spanning a mass range of roughly 39 mDa and separated by 1 mDa each. In theory, given infinite mass spec resolution, researchers could distinguish between each of these 39, allowing them to multiplex up to 39 samples in a single experiment.

Obviously, however, mass spec resolution is not infinite. Additionally, creating the distinct lysine isotopologues used in the method required time and significant expertise. Given these limitations, at the time of the original Nature Methods study, the UW-Madison researchers were able only to perform two-plex experiments.

"In the first paper we speculated about how we could make all these different versions of lysine and how we could do many more plexes," Coon said. "This paper showcases how we made the lysines and that they work like predicted – that we actually did what we projected we could do."

As Coon originally noted upon introduction of the method, NeuCode synthesizes some of the best aspects of isobaric labeling and SILAC, combining, in particular, the multiplexing capabilities of the former and the MS1 level quantitation of the latter.

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.

Because quantitation is done at the MS2 level, though, precursor interference can be a problem. With isobaric tagging, researchers isolate a precursor peptide and fragment it with MS2, causing the tag's reporter to fall off. The proportion of these reporter signals correlates to the proportions of a given peptide in the tagged 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.

SILAC – or stable isotope labeling by amino acids in cell culture – uses the incorporation of amino acids containing different isotopes to label proteomic samples for quantitative mass spec analysis. Because these differentially labeled peptides are quantitated at the MS1 level, precursor interference isn't a problem. However, incorporation of different isotopes significantly increases sample complexity, which can reduce peptide IDs if taken too far. As a result, SILAC experiments are typically limited to three-plex analysis.

NeuCode, on the other hand, allows for high multiplexing along with MS1-level quantitation. The method begins with a medium resolution (around 30,000) MS1 scan to select precursor ions for MS/MS sampling. This MS/MS measurement is also taken at medium resolution, while, at the same time, the instrument conducts a second, high-resolution MS1 scan to reveal the embedded NeuCode labels.

Because the initial MS1 scan doesn't reveal the additional complexity provided by the NeuCode labels, the method suffers less in terms of lost depth of analysis and peptide IDs than would a highly multiplexed SILAC experiment. And because the quantitation is done at the MS1 level, the method avoids isobaric labeling's precursor interference issues.

Recently, researchers – in particular Harvard University's Steven Gygi – have sought to eliminate this precursor interference problem by adding an MS3 scan to isobaric tagging experiments. In the MCP paper, Coon and his team compared NeuCode only to MS2-based isobaric tagging, but, he said, in subsequent work with MS3 methods, his group had found that while MS3 increased the method's accuracy, it resulted in a sharp drop in peptide IDs due to the increased mass spec cycle time required for the MS3 scan.

Coon noted that his team's 18-plex effort also suffered in terms of depth of proteome coverage compared to experiments using NeuCode for less ambitious plexes. The researchers quantified 603 proteins in the experiment. By way of comparison, said Anna Merrill, a researcher in Coon's group and first author on the MCP paper, in a typical two-plex SILAC experiment run on the same four-hour LC gradient, the lab would quantify in the range of 1,500 to 2,000 proteins.

"An 18-plex is not what we would do regularly," Coon said. "This is more just an exhibition of what is possible."

More typical would be a nine- or 12-plex, Merrill said, noting that in such experiments the lab generally quantifies between 1,000 and 1,500 proteins.

Coon said that he expects the level of plexing NeuCode can reasonably handle will continue to increase as mass spec instrumentation improves. In the MCP paper, the researchers used a Thermo Fisher Scientific Orbitrap Elite, which, with developer's kit modifications available from the company, can achieve resolution of 480,000.

Coon's lab also has an Orbitrap Fusion, the vendor's current flagship instrument, but, he noted, a similar developer's kit is not yet available for boosting the resolution on that model.

"My expectation is that once we get methods figured out for the Fusion, we will do better than we do with the Elite," he said.

Coon also highlighted the recent development of NeuCode labeled mice, which he and his team presented on at the American Society for Mass Spectrometry's annual meeting earlier this month. By feeding mice with NeuCode labeled media, researchers can, within two to three weeks, label animals for in vivo proteomic analysis.

The method offers an advantage over similar SILAC-labeled mice in that, while SILAC-labeled mice must have 100 percent incorporation of their labels, because such experiments compare labeled to unlabeled peptides, NeuCode-labeled mice can be only partially labeled, because the method compares levels of different labels to each other.

This, Coon said, means NeuCode mice can be labeled in a matter of weeks, as opposed to generations for SILAC mice. Additionally, researchers don't have to maintain a colony and can apply the technique to adult animals.

"It gets around a lot of the barriers with SILAC labeling," he said.

Cambridge Isotope Laboratories constructed the NeuCode labels used in the MCP paper, Coon said. He added that UW-Madison, which owns the intellectual property to the method, is currently working on a commercialization deal with an unnamed firm.

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