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SPECTROSCOPY: Two-dimensional IR method tracks proteomic fingerprints

Nov. 1, 2008

The three beams in the 2D-IR experiment include two IR (ωα and ωβ) and one visible (ωγ). The time delays of the ωβ IR pulse and visible pulse relative to the ωα IR pulse (top) are controlled by two delay stages (bottom). The lenses (L1 to L7) are all of calcium. The four-wave-mixing signal is detected by a photomultiplier after going through a series of filters (three short-pass filters at 770 nm and a 40-nm-bandwidth interference filter at 700 nm). [5]

Biological scientists have often referred to proteomics, or the study of the proteome (a word coined in 1994 from protein and genome), as the next step after the gene sequencing of the Human Genome Project. For David Klug, who chairs the Single Cell Proteomics project at Imperial College (London, England), taking that step has at times seemed more akin to “gradually sliding down the slippery slope of asking more and more involved questions while becoming increasingly dissatisfied with the available tool set.”

Proteomics presents more-complex measurement problems for investigators than genomics, because genomes remain by and large constant, while proteomes (which consist of the entire complement of proteins expressed by a genome, cell, tissue, or organism) differ from cell to cell and moment to moment, as part and parcel of normal physiological function. So while numerous methods exist for cataloguing biological entities and characterizing biological functions, one reaches a point, even in biological systems less complex than the proteome, Klug said, where measurement results “only make sense when interpreted in the full biological context.”

Klug and his colleagues at Imperial College are addressing both the lack of biological context and the slippery slope of inadequate tools by adding innovative adaptations of optical methods, such as spectroscopy, to the proteome researcher’s tool set. Currently, the most widespread method for high-throughput protein identification combines the resolving power of electrophoresis with the fingerprinting capabilities of mass spectrometry. Nuclear-magnetic-resonance (NMR) methods can also provide information on the structure and conformation of proteins.

Four-wave mixing

Klug and colleagues are adding a high-resolution optical fingerprinting method, two-dimensional (2D) IR four-wave mixing, that is broadly analogous to 2D-NMR, “but many orders of magnitude more sensitive,” Klug said. “Instead of measuring spin-spin coupling, we’re measuring vibration-vibration coupling.” This spectroscopic identification of proteins is based on unique vibrational “fingerprints” left by the side chains of each amino acid.

The experimental setup consisted of two IR optical parametric amplifiers to provide two frequency-scannable IR beams and a picosecond regenerative amplifier to provide a visible beam at 800 nm. The three beams were overlapped on the sample to produce the visible four-wave-mixing signal, which was detected using a photomultiplier (see figure). The detected signal was plotted as a function of both IR frequencies to produce 2D spectra.

Based on results achieved so far, Klug estimates that 2D-IR spectroscopic identification of between six and nine amino acids will enable unambiguous identification of 90% of human proteins. He also estimates that identification times for each protein will eventually fall between ten seconds and two minutes.^1-4

The 2D-IR electron-vibration-vibration technique may ultimately provide a relatively simple yet absolute quantification of protein levels, an increasingly important issue in many proteomic applications that is relatively difficult to achieve with mass spectrometry. But what the team has achieved so far is merely a proof of principle, Klug emphasized, and a lot of work remains to be done.

“The potential of the technique, at least in principle, is very high, but you have to imagine that this is just a short time after 2D-NMR’s invention and the technology is very clunky,” he said. “It’s going to take us a while to learn what we can really do with it. In actuality, we are investigating a whole class of potential techniques.”

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