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.1 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
spectrometer2 can directly measure the mass 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 t is gel electrophoresis3. 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.
1 DNA contains the recipes for generating or “expressing” the proteins that carry out the work of living cells.
2
An instrument used for measuring the mass of atoms and molecules with extreme precision. A sample is
ionized in a high vacuum and the ions are accelerated by strong electric fields into a field-free tube where
they drift toward a detector that produces a spike of electron current when the struck by the molecules.
Since more massive molecules drift at slower speeds than lighter molecules, the time at which the electron
signal occurs can be related very precisely to the molecular mass ..
3 Bergen County Technical Schools has a good overview of gel electrophoresis at
http://www.bergen.org/AAST/Projects/Gel/
-1-
A better way to identify proteins in a cell
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 4, 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-ionization5 (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 obtained 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 about much less than the freeelectron 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.
- VU -
4
President of Celera Genomics and leader of the private effort to map the human genome.
Desorption is the process of removing atoms and molecules that are sticking on a surface. Ionization is the
process of producing electrically charged atoms or molecules.
5
-2-