A better way to identify proteins in a cell
David F. Salisbury
Oct. 9, 2001
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
Many experts predict that proteomics will be the source of many
of the major advances in medical treatment in coming decades.
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.
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 proteinsa key step in identificationwith
unprecedented ease and sensitivity.
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
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
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.
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.
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.
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
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.
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.