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Mass Spectrometry - Accelerate Ionize Detect, Applications

job differences proteins protein molecules identify

Mass spectrometry is a technique for separating and identifying molecules based on mass. It has become an important tool for proteomics, the analysis of the whole range of proteins expressed in a cell. Mass spectrometry is used to identify proteins and to determine their amino acid sequence. It can also be used to determine if a protein has been modified by the addition of phosphate groups or sugars, for example. The technique also allows other molecules, including DNA, RNA, and sugars, to be identified or sequenced.

The use of mass spectrometry has greatly aided proteomics. Whereas DNA sequencing is simple and straightforward, protein sequencing is not. The ability to quickly and accurately identify proteins being expressed in a cell allows a range of hypotheses to be tested that cannot be approached by simply looking at DNA. For instance, it is possible with mass spectrometry to determine what proteins are expressed in cancer cells that are not expressed in healthy cells, possibly leading to further understanding of the disease and to development of drugs that target these proteins.

Data derived from mass spectrometry is usually analyzed by computer programs that search databases to help identify the analyzed protein. Such tools are the province of bioinformatics. The databases are usually located at a centralized institution and are searched via the Internet.

Proteins to be analyzed, such as those from a cell, are first separated and purified. One technique for this is two-dimensional gel electrophoresis. At the Manchester Metropolitan University in 2001, a technician prepares samples to be analyzed by a mass spectrometry machine. Combined with a database of controlled spectra, this machine can aid in the detection of anthrax spores. Individual proteins form spots on the gel, which can then be cut out individually. Chromatography can also be used. In this technique, a mixture of proteins is separated by being passed through a column containing inert beads, which slow the proteins to different extents based on their chemical properties. Unlike the two-dimensional gel method, chromatography allows continuous (versus batch) processing of cellular samples, which reduces the requirement for handling of samples and speeds up analysis.

Mass spectrometry begins by ionizing the molecules in the target sample—removing one or more electrons to give them a positive charge. Molecules must be charged so they can be accelerated. The principle is the same as that used in a television or fluorescent light bulb: Charged particles are accelerated by being pulled toward something of the opposite charge. In the mass spectrometer, the speed the molecules attain during acceleration is proportional to their mass (actually, their mass-charge ratio). By determining the speed of the molecules, researchers can calculate their mass.

Proteins are ionized in one of two common ways. The first is matrix-assisted laser desorption ionization, or MALDI. The "matrix" that is used is a crystalline structure of small organic molecules in which the protein is suspended. When excited by a laser, the protein is vaporized ("desorbed") and ionized to a +1 charge. The second method is electrospray ionization (ESI). In this process, the protein is dissolved in a solution, which is sprayed to form a fine mist (it is ionized at the same time). Evaporation of the surrounding solvent eventually leaves the protein by itself. A benefit of the solution method of ESI is that a mixture of proteins can first be separated by chromatography or capillary gel electrophoresis, and then passed on to the ionizer without additional handling, avoiding the labor-intensive two-dimensional gel method.

Following ionization, the protein is accelerated. The most common way to determine mass is with a "time-of-flight" (TOF) tube. Just as its name implies, this tube is used to determine the time of flight of the protein, allowing a simple determination of velocity (velocity = distance / time). The accelerator imparts a known amount of kinetic energy to the molecule. Since kinetic energy = 1/2(mass) (velocity)2, the determination of mass is straightforward.

Identifying Unknown Proteins.

Since several different proteins may have the same mass, simply obtaining the mass of the whole protein is not enough to identify it. However, if it is broken into a characteristic set of fragments (called peptides), and the mass of each of these is determined, it is usually possible to identify the protein based on its "peptide fingerprint."

Sequencing Peptides.

Peptides can be sequenced by generating multiple sets of fragments and analyzing the differences in masses among them. Removing a single amino acid from a peptide, for instance, will decrease its mass by a specific amount and at the same time create a new, detectable particle with the same mass. Individual amino acids can be identified by their characteristic molecular masses. Mass spectrometry has made protein sequencing much easier than it had been. The traditional method required about twelve hours to sequence a ten-amino acid peptide. Mass spectrometry can do the same job in about one second. The entire protein need not be sequenced to be identified. Often four to five amino acids are enough.

Identifying Chemical Modifications.

Chemical modifications to proteins after they are synthesized (called post-translational modifications) are important for regulation. For instance, the addition of a phosphate group (PO4) is used to turn on or turn off many enzymes. The presence of such groups can be detected by the additional weight they bring. Sugar groups can be detected in the same fashion.

Richard Robinson

Bibliography

Perkel, Jeffrey M. "Mass Spectrometry Applications for Proteomics." The Scientist 15, no. 16 (2001): 31-32.

Internet Resource

Mass Spectrometry. Richard Caprioli and Marc Sutter, eds. Vanderbilt University MassSpectrometry Research Center. <http://ms.mc.vanderbilt.edu/tutorials/ms/ms.htm>.

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