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Coon Lab Combines NeuCode Labeling with Swath-style DIA Mass Spec Analysis


NEW YORK(GenomeWeb) – Researchers at the University of Wisconsin-Madison have developed a method for combining isotopic labeling with data independent acquisition mass spec to enable quantitative multiplexing of DIA experiments.

The approach, described in a paper published last month in Analytical Chemistry, uses the neutron-encoded (NeuCode) SILAC method developed by the lab of UW-Madison researcher, and senior author on the study, Joshua Coon to get around issues that have previously limited researchers' ability to combine stable isotope labeling and DIA mass spec.

First presented in a 2013 Nature Methods paper, NeuCode 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.

To date, Coon and his colleagues have used the approach in conventional shotgun mass spec workflows. However, he told GenomeWeb, given the growing popularity of DIA mass spec, he thought the field might welcome a DIA-compatible isotopic labeling approach.

Conventional SILAC labeling uses the incorporation of amino acids containing different isotopes to label proteomic samples for quantitative mass spec analysis. However, Coon said, these isotopic peaks are far enough apart that they may not fall within the same DIA isolation window, meaning they can't be co-isolated and quantified.

Isobaric labeling methods like tandem-mass tags or iTRAQ use stable isotope tags attached to peptides of interest to enable relative or absolute quantitation of proteins. These tags fragment during MS2 to produce signals corresponding to the amount of peptide present in a sample. However, because in DIA the mass spec fragments whole swaths of precursor ions at once, the signal from the fragmented isobaric labels can't be linked back to the labeled precursor and are there uninformative.

The NeuCode labels, on the other hand, differ from one another by such small masses that they are only detected at high resolution. This means these labels are not apparent when the researchers do quantitation using an MS2 scan at medium resolution, which prevents the already complicated DIA MS2 spectra from becoming increasingly convoluted. Meanwhile, the NeuCode labels are revealed using a second, high-resolution MS2 scan.

In the standard shotgun mass spec NeuCode workflow, quantitation is done at the MS1 level. In part, Coon noted, this is necessary because the current deuterium-based NeuCode reagents don't co-elute perfectly.

"So there is a slight shift between the [Neucode label] with the most deuterium and the one without deuterium, so if you were doing DDA at MS2 you might only get a scan or two for quantitative data, and that could be skewed by this chromatographic shift," he said.

In DIA, on the other hand, because "you're profiling this whole elution period, you don't really have to worry about that issue as much," he added. "There are reagents coming out where we envision they would really not have this elution issue but we don't have those yet, so for now DIA makes a lot of sense."

Because the fragment ions in MS2 are on average lighter and less charged than the MS1 precursors, the NeuCode labels can be distinguished from one another at lower mass spec resolution.

This means that the DIA method could potentially allow for higher levels of multiplexing compared to DDA experiments run at the same resolution. Alternately, Coon said, it could allow researchers to run NeuCode DIA experiments at lower resolution, which could put the technique within reach of lower resolution machines like high-end QTOFS.

"Any Orbitrap can do this method pretty effectively, but our hope was to go beyond that and that the resolutions needed here would be accessible to high-end QTOFs," he said, adding that he believed an instrument needed to achieve in the range of 60,000 resolution to perform the DIA version of NeuCode.

For the Analytical Chemistry paper, the researchers used a Swath-style DIA approach on a Thermo Fisher Scientific Orbitrap Fusion, cycling through 25 m/z windows, said Catherine Minogue, a grad student in Coon's lab and first author on the study.

In the study, the researchers used the approach to analyze mixed ratio samples (1:1 and 10:1) of the yeast and mouse embryo myogenesis proteomes. Comparing the DIA data to that generated using the conventional DDA NeuCode technique, they found the DIA results to be comparable to those from the DDA workflow.

In the DIA yeast analysis they managed to quantify 12,986 (95.4 percent) and 11,803 (85.9 percent) of the identified peptides and established median ratios of .862 and 11.104 for the 1:1 and 10:1 mixtures, respectively. Their DDA yeast analysis quantified 13,393 (85.6 percent) and 12,112 (88.4 percent) of the identified peptides and generated ratios of .907 and 9.2 for the two mixtures.

As Coon noted, DDA analyses are still typically able to go deeper into the proteome than DIA analyses, but, DIA analyses have certain advantages – in particular, the ability to more reproducibly quantify sets of proteins across multiple samples due to the fact that the method fragments all precursor ions in a sample.

"All the things that are good about [DIA] are still there and unaffected, but we can add this ability to do multiplex quantitative labeling with isotopes," he said. "So I think that you can now envision experiments with this kind of approach that will allow one to multiplex the quantitation in Swath DIA, and there previously wasn't a good path to allow this."

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