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Seeing Proteins With Optical Spectroscopy

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Identifying and quantifying proteins can be difficult work that's fraught with limitations. When faced with a biological question that existing tools couldn't answer, Imperial College London's David Klug set about applying his background in laser technology and spectroscopy to proteomics. He and his colleagues have been developing two-dimensional infrared spectroscopy to measure and analyze proteins using optics. "It was sort of an evolution borne out of frustration, wanting to do some biology we couldn't do and an awareness that maybe doing it in this way would be interesting and different," Klug says. He is also chair of the Single Cell Proteomics project, and the technology — known as 2DIR — was developed as part of that effort.

The way that 2DIR works is analogous to two-dimensional NMR, Klug says, but instead of measuring spin-spin couplings induced by a magnet as NMR does, 2DIR measures vibrational couplings. In short, 2DIR uses infrared laser pulses to excite molecular vibration, which is then measured and used to construct a map of the vibrational couplings. "It is quite analogous to NMR in that respect. The details are, of course, completely different," he adds.

The most recent work published by Klug and his colleagues is a proof-of-principle paper showing that the fledgling technology can identify proteins. In the PNAS study, the researchers were able to identify and quantify a set of 10 proteins based on the vibrational signatures of their amino acids, having constructed a database of amino acid signatures. It is, Klug says, more sensitive than two-dimensional NMR and is quite reproducible.

"What we've shown here is that if you do things this way ... that might have some particular advantages in the future," he says. "Now what we have to do is develop [it] into a realistic, practical tool to do biology with. That's the next step."

In particular, Klug is improving the tool's throughput and is working on coupling it to a multi-dimensional separation science step. "We have to couple some of that to our equipment to separate the proteins and present them to the spectrometer in the appropriate way," he says. Also, he plans to improve the sensitivity of 2DIR to the point where it is possible to do single-cell proteomics. "You can see how that might be possible. There's principle and there's actually making it work," Klug says.

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