A better way to identify proteins in a cell

By David F. Salisbury
Oct. 9, 2001

Following the mapping of the human genome, the next "big thing" in biomedical science is likely to be proteomics: a newly coined term for identifying the structure and the role of the millions of proteins that act as the basic molecular machinery of living systems. Many experts predict that proteomics will be the source of many of the major advances in medical treatment in coming decades.

Practitioners of this new field face a number of major challenges. One of the first is coming up with fast, effective and relatively inexpensive ways of identifying these molecular movers and shakers, which are too small to see in the most powerful optical microscope.

It just so happens that the free-electron laser may provide the key to just such a system. Recent studies have shown that an FEL beam coupled with an instrument called a time-of-flight mass spectrometer can directly measure the masses of proteins—a key step in identification—with unprecedented ease and sensitivity.

One of the leading methods that biological scientists use to separate and identify different proteins is gel electrophoresis . A solution of electrically charged proteins is loaded onto one end of a long strip of special gel. When a voltage is applied between the ends of the gel, the molecules begin migrating through the gel. Lighter molecules with greater electrical charge move more rapidly than heavy molecules with fewer charges. So the molecules sort themselves into bands that contain proteins of about the same size. This approach is good for many applications, but proteomics demands more precise identifications.

For the last four years Physics Professor Richard Haglund and his students and research associates have been looking at the interactions between the FEL beam and the different ways that atoms within solid materials vibrate. In the course of these experiments, they have discovered that the tunability and short pulse length of the FEL beam can be used to selectively excite a special kind of vibration called an "anharmonic" vibration.

Solid materials vibrate in two different ways. Harmonic or lattice vibrations are like the vibrations in a slightly stretched spring: they spread rapidly throughout a material and are manifested as heat. Anharmonic vibrations, on the other hand, are strongly localized in specific atomic or molecular structures. These vibrations can be extremely energetic and last for as long as 10 to 20 trillionths of a second before transforming into harmonic vibrations.

Having found that they can stimulate anharmonic vibrations, Haglund began looking for ways to put this capability to use. Then, while reading an interview of J. Craig Venter , he learned that the most promising technology for rapidly isolating and identifying proteins uses conventional ultraviolet lasers to extract proteins from electrophoresis gels and identifies them by mass spectrometry. As he read a description of the technique, the physicist realized that the tunable beam of the FEL could do this job more easily and directly by inducing anharmonic vibrations in the gel material itself.

The major complication with using conventional lasers for protein identification is that they heat the gel/protein mixture so violently that the proteins break apart. Researchers have come up with a work-around for this problem called matrix-assisted laser desorption-ionization (MALDI) mass spectrometry (MS). They do so by adding another material, called a matrix, to the protein-gel mixture. The matrix absorbs much of the energy in the laser beam and so moderates the heating rate so that enough intact proteins are released and ionized to be identified. The addition of the matrix material is the most complicated and time-consuming step in the entire process. Also, many matrix materials are acidic and induce chemical changes in the proteins or alter their delicate conformation.

Michelle Baltz-Knorr, one of Haglund's graduate students, was interested in exploring how the FEL beam interacts with the gel/protein system. She acquired samples of electrophoresis gel from colleagues in Molecular Biology and began studying what happened when she irradiated it with different wavelengths of infrared laser light. At a wavelength of 5.9 microns, she hit pay dirt. She discovered that the gel molecules began loosening up and ejecting intact protein ions. Using a mass spectrometer on the FEL beam line, she and Haglund demonstrated that they can identify proteins in electrophoresis gels without going through the time-consuming sample-preparation stage in conventional MALDI-MS. They have named the new process RIR-MALDI, for Resonant InfraRed MALDI.

The University has applied for a patent on the process and discussions are underway about developing a special-purpose, solid-state laser that would cost much less than the free-electron laser, but duplicate the special features of the FEL beam required for this purpose.

The scientists believe that they can take this process even further. Baltz-Knorr has run additional experiments that show it is possible to identify proteins encased in ice, rather than gel. The next challenge will be to demonstrate that this method can identify a protein in extremely low concentrations, as small as 100 to 1000 proteins in a trillionth of a liter of water. This would open up the possibility of identifying proteins directly within individual cells.

Overview of gel electrophoresis:
http://www.bergen.org/AAST/Projects/Gel/