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No Optical Illusion: UK Researchers Developing New Proteomics Platform

Researchers in the UK are developing an optics-based technology that they say could provide a new method for proteomics and protein analysis. The system, based on two-dimensional infrared spectroscopy, is still in embryonic stage and in need of rigorous testing, but according to the corresponding author of an article describing it, the platform has “unique potential.”
“There is no existing method for doing this,” David Klug, professor of chemical biophysics at the Imperial College of London, told ProteoMonitor this week. “This is the first time that it’s been done that you can identify a protein optically in a generic sense.”
Klug is also chair of Single Cell Proteomics, a project begun in 2006 to analyze and measure proteins found in cells. The 2DIR technology was developed as part of the project.
While other approaches to developing an optics-based system have been attempted and shown “significant potential,” they have suffered from “overcongested spectra, which makes feature assignment and quantification highly problematic,” Klug and his co-authors said in an article published Oct. 1 in the Proceedings of the National Academy of Sciences, Early Edition.
Their platform, combining 2DIR along with electronic vibration-vibration to “decongest 2DIR spectra,” is in “primitive” form, Klug said, and is not meant to compete with — much less replace — either mass spectrometers or microarrays, the two main technologies used for protein analysis.
But, Klug said, while mass specs and microarray technology have made “fantastic progress” allowing researchers to find things they couldn’t imagine just a few years ago, they are “imperfect” and different technology could help fill in information gaps that current instruments are unable to address.
“We didn’t set out to compete with microarrays or mass spectrometry,” he said. “There’s no point. … What you have to do is you have to look and see what the strengths are of what you’re doing and see where and when they overlap relative to the weakness of the other methods.”
In earlier work, he and his colleagues showed how such a platform can be used for peptide analysis by quantifying relative amino acid levels for short peptides. Primary, secondary, and tertiary structural effects could prevent such measurements on proteins, they said, but in the work described in the PNAS article, they show that “protein identification can be performed by using spectroscopically determined amino acid content, relative to an internal reference.”
The platform they’ve developed, they added, “can be used to differentiate and identify proteins and to measure absolute protein quantities. We also demonstrate that the sensitivity and throughput of our EVV 2DIR apparatus is sufficient for this method to be considered for use as a real proteomic tool.” 
The current work on the platform is two-fold, Klug said: to develop it into a working biological platform and to find applications for the technology.

“This is the first time that it’s been done that you can identify a protein optically in a generic sense.”

On the first task, Klug said, the researchers are coupling the EVV 2DIR system to multi-dimensional methods such as multiple-step liquid chromatography followed by electrophoresis, a step used also in mass spectrometry-based methods.
“The difference is that we’re using intact proteins … rather than peptide fragments,” which could prove useful for obtaining structural information about the proteins, he said. 2DIR, in principle, contains structural information, though no one has yet been able to get such distance information reliably from a 2DIR spectrum.
“We and others are working on it, however, and this might prove useful in a high-throughput context,” Klug said. “Even if whole structures cannot be solved, some distance relationships may be measurable.”
On the second task, the researchers have identified absolute quantification as one area in which their platform potentially could work very well. While absolute quantification is becoming a crucial part of many proteomic experiments, mass spectrometry is not especially good for such work, the authors said in their article.
By comparison, absolute quantification on the EVV 2DIR platform can be done “very easily and conveniently,” Klug said. And because it can be done on the platform without the use of labels, it could have use especially for clinical purposes.
“The labeling techniques in MS have made great progress and are very powerful, but it’s a bit hard if you’ve got clinical samples, “ Klug said. “You just can’t label them. You can’t get patients to take isotopes.”
Preliminary results suggest that EVV 2DIR may also be useful for analysis of post-translational modifications. “The spectroscopy naturally just picks up signatures of chemical groups … and it does that in such a way that a spectral space can be found, or that chemical group can be seen away from all the other features,” Klug said. He and his colleagues have been able to pick up phosphorylation “pretty easily,” in their work and are further testing the use of the system for detecting that and other forms of PTMs.
“The technology is quite clunky … so I think it’s going to take us a while to figure out what the [unique selling propositions] are, what the clear applications are. We have to try a range of things,” Klug said.
In addition, the research team is working to improve the sensitivity of the platform, coupling it to the multi-dimensional separation systems, and improving the imaging. Members of the team are also working on developing the bioinformatics for the platform, as the bioinformatics for EVV 2DIR are “completely different” from that for fragment-based analysis, Klug said.
Optical Methods
The EVV 2DIR platform comes as the result of work being done under the framework of the Single Cell Proteomics project, started two years ago with £5 million ($9 million) in funding from the Energy and Physical Sciences Research Council in the UK. As part of that the initiative, researchers went about developing new technologies for protein analysis [See PM 02/15/07].
The idea for the EVV 2DIR system, however, had been germinating in Klug’s brain for at least 15 years, he said. While studying natural photosynthetic systems, mostly membrane protein systems, he obtained results on the mechanisms of energy electron transfer in these systems. In order to interpret the results, one needed to put them in the “the full physiological context of the biology because these are highly regulated reactions [and] there are multiple forms of regulation that go on in the higher plants to control these,” he said.
To make a quantitative model of the bigger picture, “I wanted to see what biological, biochemical information there was about the plasticity of the membrane, the extent of association of the different proteins,” he said. “And the information just wasn’t there.”
Poking around proteomics to find a solution, he discovered that there were “no optical approaches under development for protein analysis at all. … And I thought it would be very interesting to see what we could do using optical methods, and because I was interested in multi-dimensional spectroscopy, which was just starting to emerge as a subject at around the same time, I thought it would be very interesting to develop a method in this particular direction and see what we could make of it.”
In addition to work on the EVV 2DIR, he and his colleagues also are developing a technology based on optically trapped smart droplet microtools for the study of membrane proteins from a single cell. Their method, they said in an article published during the summer, would facilitate the high-content analysis and quantification of species.
In the meantime, they are in discussions with proteomics scientists for their input on the EVV 2DIR system.
“I think with the proteomics community, we just have to sit down and … try to learn where it’s particularly good and where we can make a real impact on which particular bits of biology,” Klug said. “And that means we have to do some real biology with it,” a step that he said he expects to happen within 12 months. At that point, he added, the platform should be ready for development as an alpha tool as a first draft for an instrument that could be made commercially available.

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