Proteins are large, complex molecules that carry out the tasks of life. They direct our
bodies' activities, organize our thoughts, and defend us against infection, keeping us
healthy. But in their mutant forms and as coats on disease-causing microbes, proteins
can help make us ill and threaten our health.
Each protein is initially formed as a string of amino acids whose identity and order are
dictated by a gene according to the sequence of its DNA bases. The gene's
instructions—carried by messenger RNA—also call for this string to be folded into a
three-dimensional molecule that has an intricate shape, ranging from a saucer to a
dumbbell to a corkscrew.
The amino-acid composition and sequence, as well as the molecular weight, of a protein
produced by a certain type of bacteria are different from those of the protein forming
the coat of a particular virus. Such a coat enables this molecular terrorist to break
through the protective membrane of a cell and command it to produce more virus. Each
type of microbe produces unique proteins, providing a characteristic protein signature
and allowing identification of the microorganism. Thus, if the signature proteins in
anthrax spores and botulism toxins could be accurately detected, it would be possible to
provide an early warning about the proximity of biological warfare weapons.
The ability to identify proteins is also important because it allows researchers to
determine whether an organism has a genetic disease. A genetic disease is often caused
by a mutant protein, which has a composition slightly different from that of the normal
protein it replaces.
One of the most powerful tools for detecting and identifying proteins is mass
spectrometry, a technique that has been improved and used for a variety of research
projects for many years at ORNL. A mass spectrometer sorts out charged particles
according to their masses, allowing analysis of the elemental composition of complex
molecules. A mass spectrometer produces a spectrum consisting of peaks and valleys
that indicate the identity and number of different atoms making up the molecule being
The mass spectrometer is an ideal instrument for identifying amino acids—the building
blocks of proteins—and determining the order in which they are arranged. The mass
difference, or distance in atomic mass units between the peaks along the spectrum,
allows each amino acid (e.g., alinine, arsenine, glycine, or lysine—four of the 20
possible amino acids) to be identified.
New ionization methods aid protein analysis
With the discovery in the past decade of two powerful methods that can be used to
produce ions for analysis in a quadrupole ion trap mass spectrometer, progress in
biological mass spectrometry has been nothing short of revolutionary. Mass
spectrometry has become, in just a few years, an important tool for protein
identification, peptide sequencing, identification and location of post-translational
modifications of proteins, analysis of modified deoxyribonucleic and ribonucleic acids
(DNA and RNA), and many other biological applications. These advances have become
possible only through the capability to form ions from large biomolecules and through
the research community's growing understanding of the chemistry of biological ions.
During the mid-1980s, the quadrupole ion trap was
beginning to emerge as a mass analyzer with
interesting characteristics for tandem mass
spectrometry. The quadrupole ion trap operates on
the principle that ions can be stored within an
oscillating electric field. With appropriately shaped
electrodes, an oscillating electric field (usually a
quadrupole field or a variation thereof) can be
created that stores ions in three dimensions.
Furthermore, the amplitude (strength) of the electric
field can be changed so that ions of different
mass-to-charge ratios are ejected from the ion trap
and into a detector. In this way, the ion trap serves
as a mass spectrometer.
In the latter part of the 1980s, the Organic Mass
Spectrometry Group in ORNL's Chemical and
Analytical Sciences Division (CASD)—one of the
leading groups in the world today in this area of
research—began to study electrospray ionization. It
is one of the important new ionization methods for
mass spectrometry because it forms gaseous ions
from polar and nonvolatile molecules in solution,
without the addition of heat. Gary Van Berkel, Scott
McLuckey, and Gary Glish, all of CASD, were the
first to couple the electrospray ionization technique
with the ion trap mass spectrometer.
"In electrospray, a tiny drop of protein-containing solution is injected into a thin glass
needle," says CASD's Jim Stephenson. "The needle is held at a potential of several
thousand volts, providing energy and adding multiple charges to the protein. As the
solution leaves the needle, it evaporates, forming a fine mist of charged droplets from
which ions emerge. These ions in a gaseous state are introduced to the mass
Another relatively new ionization method used at ORNL to analyze biological samples is
matrix-assisted laser desorption (MALDI) mass spectrometry. For protein analysis, a
protein is dissolved in solution and added to a matrix. The mixture is placed on a probe
tip, which is illuminated by an ultraviolet light beam from a nitrogen laser. The laser
beam desorbs the protein off the surface of the probe tip. Protons are then transferred
between the protein and matrix, leaving the protein as a negatively charged ion.
Biological Applications Of Mass Spectrometry
Since coming to ORNL in 1995 as a postdoctoral researcher, Stephenson, now a staff
scientist in CASD's Organic Mass Spectrometry Group, has been participating in
biological research using mass spectrometry. He has been working with other CASD
researchers, particularly Keiji Asano, Doug Goeringer, Bob Hettich, Greg Hurst, Rose
Ramsey, Gary Van Berkel, and Michelle Buchanan, now director of CASD.
"We use mass spectrometry to identify the characteristic protein signatures of various
bacteria and viruses, such as the tobacco mosaic virus," he says. "We extract proteins
from Escherichia coli bacteria cells and look for proteins unique to these organisms.
We can identify the signature proteins in anthrax spores and botulism toxins."
Using mass spectrometry,
Stephenson and his ORNL
colleagues obtain two important
pieces of information that allow
them to identify proteins. First,
they get the molecular weight of
the protein. Second, by
measuring the distances
between the peaks in the
spectrum, they can figure out
which amino acids are present
and in what order to get the
"sequence tag." They also can
deduce the amino-acid
sequence by the way the
protein falls apart when charges and energy are added to it.
"With this information, we can go to the protein sequence database and identify the
protein," Stephenson says. "For example, we might find six tobacco mosaic virus
proteins in the database that have the same sequence tag but different molecular
weights. Only one protein in the database matches the molecular weight and sequence
tag we found."
Stephenson and his colleagues have been analyzing the disease state of mutant mice in a
project with mouse geneticist Gene Rinchik of ORNL's Life Sciences Division. For this
experiment, Rinchik exposes male mice to the powerful chemical mutagen
ethylnitrosourea (ENU) and mates them with female mice from the same strain. As a
result, many different mutations are produced in otherwise identical offspring because
ENU can alter a single base pair in a gene. Stephenson and Rinchik are interested in
using the mass spectrometer to detect mutant proteins that result from these inherited
genetic changes. As a proof of principle, they are initially looking for mice born with a
mutant hemoglobin protein.
Red blood cells carry oxygen to all parts of the body
by using a protein called hemoglobin. A small
sample of blood is easy to obtain from a large
number of mice, so hemoglobin is a good model to
test whether or not the mass spectrometer could
find small inherited protein changes in the progeny
of mice treated with the ENU mutagen. Rinchik
was able to obtain a sample of blood with known
mutant hemoglobins from Ray Popp, retired from
ORNL's former Biology Division, and Stephenson
found that the mass spectrometer could easily
identify the changes. "Now," Stephenson says, "we
can use mass spectrometry on blood samples from
the offspring of mutagen-treated mice to detect new
mutations. If we are successful in recognizing
hemoglobin protein variants, we can use the
technique to identify new, inherited mutations in
any protein, increasing the efficiency and reducing
the cost of finding new inherited variants in mice."
The ORNL group also uses mass spectrometry to
detect post-translational modification (PTM)
proteins. In a cell, a protein can be modified in
different ways by other proteins or by exposure to a pollutant. For example, in a
process called phosphorylation, a phosphokinase enzyme can attach a phosphate to a
protein to activate it or remove a phosphate to inactivate the protein. Mass spectrometry
can be used to confirm the presence or absence of a phosphate in a protein.
In addition, Stephenson and his colleagues are using electrospray ionization mass
spectrometry (ESI/MS) to help determine the three-dimensional structure of proteins.
Using a cross-linking chemical of a known length that attaches between two neighboring
lysines in a polypeptide chain, the group can measure the molecular distance between
these amino acids. This information is of value to the Computational Protein Structure
Group, led by Ying Xu, which is part of LSD's Computational Biology Section. This
group uses a protein-threading computer model to predict the structure of proteins. (See
Protein Prediction Tool Has Good Prospects.)
Speeding Up Protein Identification
In a project supported by internal funding from the Laboratory Directed Research and
Development Program, Stephenson and his colleagues further developed ESI/MS so
that it could analyze proteins much faster than the conventional method he describes
"Traditionally, we take a purified protein and break it into smaller pieces by digesting it
with a proteolytic enzyme that selectively cleaves the protein at specified amino acid
sites. The resulting products from the proteolytic digestion are then separated on a liquid
chromatography column and then are transferred to the mass spectrometer directly.
Sequence tags are then generated by adding energy to the protein pieces via collisions
with helium atoms. These protein pieces fall apart into the individual amino acids of the
protein. From these data we can figure out the sequence of the sequence tag and
identify the protein by checking the sequence tag and molecular weight against protein
data in the database. This approach takes about a day."
By making improvements in the ESI/MS technique and eliminating liquid
chromatography, Stephenson and his associates could analyze a single protein in just a
few minutes, not a day. "The problem then was how to use ESI/MS to identify many
different proteins in a complex mixture at once. You could present one protein at a time
to the mass spectrometer after separating the protein mixture by liquid chromatography
or gel electrophoresis. But this approach is time consuming. So we designed an ion-ion
reaction instrument to separate out a target protein or allow the mass spectrometer to
look at one protein at a time."
Stephenson and Ben Cargile, a former graduate student at the University of Tennessee,
developed a protein identification algorithm based on their discovery of how intact
proteins fall apart when energy is added. This algorithm can be used to take
sequence-specific data from intact proteins and identify them through a database search.
Speeding up protein identification and the collection of information on the compositional
differences between normal and mutant proteins and the measurements of distances
between protein building blocks could lead to the rapid development of more effective
therapeutic drugs. ORNL's capabilities in combining computational analysis with data
obtained by ORNL's Organic Mass Spectrometry Group could result in increased
protection and improvement of human health.
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