NEW YORK (GenomeWeb) – University of Wisconsin-Madison researcher Josh Coon has applied his lab's NeuCode protein labeling technique to in vivo use in mice.
Detailed in a paper published last month in Cell Reports, the approach could prove a faster and less expensive alternative to in vivo SILAC labeling while also offering higher multiplexing capabilities, Coon told GenomeWeb.
Developed by Coon's lab in 2013, NeuCode is a metabolic labeling method that uses the 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 label proteomic samples.
In their initial study, Coon and his colleagues used different isotopes of carbon, hydrogen, nitrogen, and oxygen to work out 39 different isotopologues for lysine separated by 1 mDa each. At the time, Coon noted that this, in theory, could allow for multiplexing of up to 39 samples in a single experiment, provided high enough mass spec resolution.
In practice, the group has typically used the method for nine or 12-plex experiments, in which it can generally quantify in the range of 1,000 to 2,000 proteins. In 2014, Coon and his colleagues published a paper in Molecular & Cellular Proteomics in which they combined NeuCode with dimethyl labeling to multiplex 18 samples in a single mass spec experiment.
More recently, the researchers have looked to apply the NeuCode technique to in vivo labeling in mice. They first presented on the idea at the 2014 American Society for Mass Spectrometry annual meeting. Last month's Cell Reports paper marks the first published demonstration of the technique, Coon said.
Currently, mouse SILAC, or SILAM, is the most commonly used approach for metabolically labeling mice. SILAC — or stable isotope labeling by amino acids in cell culture — uses the incorporation of amino acids containing heavy isotopes to label proteomic samples for quantitative mass spec analysis.
The technique is very limited in terms of multiplexing, however, Coon noted. For instance, he said, in a traditional SILAM experiment, researchers use the heavy labeled SILAC mouse as a reference sample to which they compare the experimental mouse.
"So, you are running two samples at once, but if you wanted to compare two experimental conditions you basically have to do two experiments," he said. "Condition one compared to your SILAC reference mouse, and then experimental mouse two compared to your SILAC reference mouse. You are typically indirectly comparing your experimental animals through this reference mouse."
NeuCode's higher multiplexing capability means researchers can directly compare a number of different experimental conditions, Coon said. In the Cell Reports study, he and his colleagues multiplexed four mice at once. Currently, he said, that is probably the technique's upper limit in terms of labeled mice. However, he noted, as mass spec resolution improves, multiplexing should go up.
"I think that for seven-plex it is pretty straightforward to envision how we get there, and I think as resolution improves we will be able to go even higher," he said. "It all hinges on the resolution of the mass spectrometer you are using."
The researchers used a Thermo Fisher Orbitrap Elite for the study, but they have since begun using newer, higher-resolution instruments including the Orbitrap Fusion Lumos.
In addition to improved multiplexing, the technique also offers potential savings in terms of cost and time compared to SILAM, Coon said. This is because, unlike SILAM, the NeuCode-based method doesn't require that animals be completely labeled.
Because SILAM experiments are comparing heavy-labeled animals to unlabeled animals, it is essential that the labeled animals fully incorporate the labels. Otherwise, you will have unlabeled signal from both the labeled and unlabeled mouse, affecting the accuracy of your measurements.
Ideally, Coon said, SILAM experiments need around 95 percent incorporation of the labeled amino acids. Achieving this is difficult and expensive, though, due to the varying rates at which different tissues and proteins will incorporate the labeled amino acids.
"For instance, the proteins in the liver will incorporate [the labeled amino acids] faster on average than those in the brain or muscle," Coon said. "All these tissues have different rates of proteins turnover, and therefore different rates of incorporation, and even within a tissue you would see a pretty broad range of incorporation rates where some proteins turn over frequently and they would incorporate the label more quickly than say a different protein."
To get complete labeling, SILAM mice are often fed labeled food for as many as three generations, which means producing such animals takes a number months and large amount of labeled material.
The NeuCode method, on the other hand, is not comparing labeled to unlabeled animals but, rather, animals containing different labels. This gave Coon and his researchers the idea that so long as the animals were incorporating the labels at the same rate, they might not need to completely incorporate the labels for the experiment to work.
"And it turns out that is true," he said. "We can take adult mice and feed them [labeled food] for as little as a few days and in some tissues we can get 50 percent incorporation and do good quantitation."
In general, Coon said, he and his colleagues found that they could label most tissues with two to three weeks of feeding the NeuCode-labeled material.
"So now we are talking about a very different experiment in that we can compare multiple animals at once, and all I have to do is feed an animal a special diet for two to three weeks," he said. He added that while he could not say how much a NeuCode-labeled mouse would cost, he expected it would be significantly less than the price for SILAC-labeled mice, which run around $6,000.
While SILAC-labeled mice are "a technique that maybe pharma can afford," it has remained out of reach for many researchers, Coon said, adding that he expected the NeuCode-based method would prove more accessible. "Now you can work with your own animals and compare them directly with very short labeling times."
Coon's lab has been working with Cambridge Isotope Laboratories on developing the NeuCode reagents as commercial products. CIL will be commercializing the NeuCode mouse feed and has entered a partnership with Thermo Fisher Scientific under which that company will provide NeuCode reagents for cell culture experiments. Coon said he expected these reagents would be available within the next year.