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Protein identification by mass spectrometry

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 analyzed.

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 spectrometer."

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 below:

"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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