This story has been updated to correct an earlier version that identified Steven Gygi as an MIT researcher. He is a researcher at Harvard University.
University of Wisconsin-Madison researchers have developed a new labeling approach that could enable significantly higher multiplexing in mass spec proteomics experiments.
Detailed in a paper published this week in Nature Methods, the technique, called NeuCode, uses differences in the nuclear binding energy of different isotopes to label amino acids. According to Josh Coon, professor of chemistry and biomolecular chemistry at UW-Madison and leader of the effort, the new method brings together benefits of SILAC labeling and isobaric tagging.
Nuclear binding energy is the energy needed to break a nucleus up into its component nucleons, and because every isotope has a unique nuclear binding energy, these differences can be used to distinguish between them.
Last year, two groups – one led by Harvard University researcher Steven Gygi and another by researchers from chemical proteomics firm Cellzome (since acquired by GlaxoSmithKline) – applied this phenomenon to isobaric tagging, swapping the tags' 12C atoms for 13C atoms and 15N for 14N to produce a 6-mDa mass shift that upped the reagents' multiplexing capability from six-plex to eight-plex.
In their study released this week, Coon and his team built on this work by incorporating a number of different isotope combinations into lysine molecules, which they then 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.
Given infinite mass spec resolution, Coon told ProteoMonitor, researchers could distinguish between each of these 39, allowing them to multiplex up to 39 samples in a single experiment. Because mass spec resolution isn't infinite, however, the UW-Madison team maxed out at six-plex analysis, running the experiment at 240,000 resolution on a Thermo Fisher Scientific Orbitrap Elite.
Since then, the researchers have managed to achieve 12-plex analysis using the technique at 500,000 resolution on a specially modified Orbitrap Elite. And, Coon said, he believes that at 1 million resolution – a level that Orbitrap Elites have, under certain circumstances, managed to achieve – the technique will enable 21-plex analysis.
The method, Coon noted, 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.
These reagents, which 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, enable researchers to multiplex up to eight samples.
However, because quantitation is done at the MS2 level, 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, with the proportion of these reporter signals correlating 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, potentially enables high multiplexing along with MS1-level quantitation.
The approach "compresses all the [isotopic] information into tens of milliDaltons, so it's only revealed when you crank up the [mass spec's] resolution," Coon said. "We think we can go to a really high level of plexing without affecting the number of identifications or increasing the [sample] complexity."
In the Nature Methods paper, the researchers benchmarked the NeuCode technique against conventional SILAC, analyzing mouse C2C12 myoblasts and their differentiation to myotubes. Using NeuCode they identified 5,747 quantifiable peptides versus 3,400 via standard SILAC.
Harvard's Gygi, who was not involved in the NeuCode research, called the approach a "very clever idea," telling ProteoMonitor via email that it provides a route toward higher SILAC-style multiplexing.
He noted, however, that additional developments would be required for it to become a widely useful tool. Specifically, he said, instrument vendors would have to embrace the idea and develop machines aimed at proteomics researchers that offered the extremely high sensitivity needed for the technique.
In addition, he said, creating the various lysine isotopologues would "require some sophisticated chemistry partner."
Indeed, Coon said that the main reason the researchers had not yet pushed the technique beyond six-plex was a lack of additional lysine isotopologues. He and his team are currently working to develop additional reagents. According to a release from UW-Madison, the researchers have filed a patent on the technique, the rights to which have been assigned to the Wisconsin Alumni Research Foundation.
Regarding the method's reliance on high-resolution mass spec, Coon said that, aside from FT-ICR machines, the Orbitrap mass analyzers are the only other commonly used commercial systems that offer the necessary resolution.
Time-of-flight systems "just don't get enough resolution," he said. "So I don't think with a TOF there's going to be much ability to use [NeuCode] at least the way we are currently pursuing it.
Even the standard model Orbitrap Elite tops out at 240,000 resolution, which is enough for six-plex NeuCode, but far short of what is required to achieve the technique's potential. However, Coon noted, with a few software modifications these instruments can reach 480,000 resolution. And, as Alexander Makarov, inventor of the Orbitrap and director of research for life science mass spectrometry at Thermo Fisher, demonstrated in a paper last year in the International Journal of Mass Spectrometry, modified Orbitrap Elites can under certain conditions reach 1 million resolution.
"I don't think there's ever been a market for [such high resolution]," Coon said. "The shotgun proteomics community has never really needed it. But this method presents a real opportunity if you can achieve [such resolution.] So hopefully we'll see efforts to make it more routine."
"Clearly Orbitraps can deliver [such a level of resolution], but I just don't think [Thermo Fisher has] marketed it because the market has been limited," he added.
Thermo Fisher declined to comment on the NeuCode technology or the company's potential interest in it.
In addition to developing the method for SILAC-style metabolic labeling, Coon and his team are also working to develop it as a chemical tagging reagent. In addition to enabling researchers to use it in samples, such as human tissue, where metabolic labeling is unfeasible, a chemical tagging version could prove amenable to higher multiplexing and lower mass spec resolution, he said.
This is because these tags would not have to be amino acids like lysine, and so wouldn't be limited by such molecules' inherent structures.
"With a lysine molecule we have only so many atoms to play with," Coon said. "But in a [chemical] tag, you could design it however you want. So you could have it be a little bit bigger and have the isotopologues spaced exactly as you want."
"So I would expect the tag work to offer more plexing and lower resolution demands," he said.