Mass Spectrometry

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.

Ionize, Accelerate, Detect

Proteins to be analyzed, such as those from a cell, are first separated and purified. One technique for this is two-dimensional gel electrophoresis . 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.

Applications

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.

see also Bioinformatics; HPLC: High-Performance Liquid Chromatography; Internet; Post-Translational Control; Proteins; Proteomics.

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>.

Mass Spectrometry

Mass spectrometry is an instrumental method of obtaining structure and mass information about either molecules or atoms by generating ionized particles and then accelerating them in a curved path through a magnetic field. Heavier particles are more difficult for the magnetic field to deflect around the curve, and thus travel in a straighter path than lighter particles. Consequently, by the time the particles reach the detector, a mixture of ions will have separated into groups by mass (or more specifically the mass-to-charge ratio of the individually weighted ions.) The ions are produced from neutral molecules and atoms by stressing them with some form of energy to knock off electrons. In the case of molecules, fragmentation as well as ionization usually occurs.

The primary reasons for using a mass spectrometry are: determination of isotopic composition of elements within a compound; identification of a unknown compound by its molecular mass or by its fragmentation; determination of a compound structure by its fragmentation; research into the characteristics of ions within a vacuum (what is called gas phase ion chemistry); determination of the amount of a compound; and determination of other chemical, physical, and biological properties of a compound.

Each type of molecule breaks up in a characteristic manner, so a skilled observer can interpret a mass spectrum much like an archaeologist can reconstruct an entire skeleton from bone fragments. A mass spectrum can help establish values for ionization energy (the amount of energy it takes to remove an electron from a neutral atom or molecule) and molecular or atomic mass for unknown substances. The extremely high sensitivity of mass spectrometry makes it indispensable for analyzing trace quantities of substances, so it is widely used in environmental, pharmaceutical, forensic, flavor, and fragrance analysis. The petroleum industry has used mass spectrometry for decades to analyze hydrocarbons.

The basic principle underlying mass spectrometry was formulated by English physicist Joseph John (J.J.) Thomson (1856–1940), he discoverer of the electron, early in the twentieth century. Working with cathode ray tubes, he was able to separate two types of particles, each with a slightly different mass, from a beam of neon ions, thereby proving the existence of isotopes. (Isotopes are atoms of the same element that have slightly different atomic masses due to the presence of differing numbers of neutrons in the nucleus.) The first mass spectrometers were built in 1919 by British physicist Francis William Aston (1877–1945) and Canadian-American Arthur Jeffrey Dempster (1886–1950).

There are five major parts to a mass spectrometer: the inlet, the ionization chamber, the mass analyzer, the detector, and the electronic readout device. The sample to be analyzed enters the instrument through the inlet, usually as a gas, although a solid can be analyzed if it is sufficiently volatile to give off at least some gaseous molecules. In the ionization chamber, the sample is ionized and fragmented. This can be accomplished in many ways—electron bombardment, chemical ionization, laser ionization, electric field ionization—and the choice is usually based on how much the analyst wants the molecule to fragment. A milder ionization (lower electric field strength, less vigorous chemical reaction) will leave many more molecules intact, whereas a stronger ionization will produce more fragments. In the mass analyzer, the particles are separated into groups by mass, and then the detector measures the mass-to-charge ratio for each group of fragments. Finally, a readout device—usually a computer—records the data.

Mass spectrometers are often used in combination with other instruments. Since a mass spectrometer is an identification instrument, it is often paired with a separation instrument like a chromatograph. Sometimes two mass spectrometers are paired, so that a mild ionization method can be followed by a more vigorous ionization of the individual fragments.

Mass spectrometers have often gone into space. Two mass spectrometers were taken aboard the Viking 1 and 2 spacecraft launched by the United States in 1975–1976. More recently, in 2005, the Cassini–Huygens spacecraft used a Gas Chromatography-Mass Spectrometry (GC-MS) instrument onboard the Huygens probe to analyze the atmosphere of Titan, Saturn’s largest moon, when it descended through its atmosphere.

See also Spectroscopy.

Mass spectrometry

Mass spectrometry is a method for finding out the mass of particles contained in a sample and, thereby, for identifying what those particles are. A typical application of mass spectrometry is the identification of small amounts of materials found at a crime scene. Forensic (crime) scientists can use this method to identify amounts of a material too small to be identified by other means.

The basic principle on which mass spectrometry operates is that a stream of charged particles is deflected by a magnetic field. The amount of the deflection depends on the mass and the charge on the particles in the stream.

Structure of the mass spectrometer

A mass spectrometer (or mass spectrograph) consists of three essential parts: the ionization chamber, the deflection chamber, and the detector. The ionization chamber is a region in which atoms of the unknown material are excited so as to make them lose electrons. Sometimes the energy needed for exciting the atoms is obtained simply by heating the sample. When atoms are excited, they lose electrons and become positively charged particles known as ions.

Ions produced in the ionization chamber leave that chamber and pass into the deflection chamber. Their movement is controlled by an electric field whose positive charge repels the ions from the ionization chamber and whose negative charge attracts them to the deflection chamber.

The deflection chamber is surrounded by a strong magnetic field. As the stream of positive ions passes through the deflection chamber, they are deflected by the magnetic field. Instead of traveling in a straight path through the chamber, they follow a curved path. The degree to which their path curves is determined by the mass and charge on the positive ions. Heavier ions are not deflected very much from a straight line, while lighter ions are deflected to a greater extent.

When the positive ions leave the deflection chamber, they collide with a photographic plate or some similar material in the detector. The detector shows the extent to which particles in the unknown sample were deflected from a straight line and, therefore, the mass and charge of those particles. Since every element and every atom has a distinctive mass and charge, an observer can tell what atoms were present in the sample just by reading the record produced in the detector.

Credit for the invention of the mass spectrometer is usually given to British chemist Francis William Aston (1877–1945). Aston made a rather remarkable discovery during his first research with the mass spectrograph. When he passed a sample of pure neon gas through the instrument, he found that two separate spots showed up in the detector. The two distinct spots meant that the neon gas contained atoms of two different masses.

Aston interpreted this discovery to mean that two different kinds of neon atoms exist. Both atoms must have the same number of protons, since all forms of neon always contain the same number of protons. But the two kinds of neon atoms must have a different number of neutrons and, therefore, different atomic masses. Aston's work was the first experimental proof for the existence of isotopes, forms of the same atom that have the same number of protons but different numbers of neutrons.

[See also Cathode-ray tube; Isotope ]

Mass spectrometry

Mass spectrometry is an instrumental method of obtaining structure and mass information about either molecules or atoms by generating ionized particles and then accelerating them in a curved path through a magnetic field. Heavier particles are more difficult for the magnetic field to deflect around the curve , and thus travel in a straighter path than lighter particles. Consequently, by the time the particles reach the detector, a mixture of ions will have separated into groups by mass (or more specifically the mass-to-charge ratio of the individually weighted ions.) The ions are produced from neutral molecules and atoms by stressing them with some form of energy to knock off electrons. In the case of molecules, fragmentation as well as ionization usually occurs. Each type of molecule breaks up in a characteristic manner, so a skilled observer can interpret a mass spectrum much like an archaeologist can reconstruct an entire skeleton from bone fragments. A mass spectrum can help establish values for ionization energy (the amount of energy it takes to remove an electron from a neutral atom or molecule) and molecular or atomic mass for unknown substances. The extremely high sensitivity of mass spectrometry makes it indispensable for analyzing trace quantities of substances, so it is widely used in environmental, pharmaceutical, forensic, flavor, and fragrance analysis. The petroleum industry has used mass spectrometry for decades to analyze hydrocarbons.

The basic principle underlying mass spectrometry was formulated by J. J. Thomson (the discoverer of the electron) early in the century. Working with cathode ray tubes, he was able to separate two types of particles, each with a slightly different mass, from a beam of neon ions, thereby proving the existence of isotopes. (Isotopes are atoms of the same element that have slightly different atomic masses due to the presence of differing numbers of neutrons in the nucleus.) The first mass spectrometers were built in 1919 by F. W. Aston and A. J. Dempster.

There are five major parts to a mass spectrometer: the inlet, the ionization chamber, the mass analyzer, the detector, and the electronic readout device. The sample to be analyzed enters the instrument through the inlet, usually as a gas, although a solid can be analyzed if it is sufficiently volatile to give off at least some gaseous molecules. In the ionization chamber, the sample is ionized and fragmented. This can be accomplished in many ways—electron bombardment, chemical ionization, laser ionization, electric field ionization—and the choice is usually based on how much the analyst wants the molecule to fragment. A milder ionization (lower electric field strength, less vigorous chemical reaction) will leave many more molecules intact, whereas a stronger ionization will produce more fragments. In the mass analyzer, the particles are separated into groups by mass, and then the detector measures the mass-to-charge ratio for each group of fragments. Finally, a readout device—usually a computer—records the data.

Mass spectrometers are often used in combination with other instruments. Since a mass spectrometer is an identification instrument, it is often paired with a separation instrument like a chromatograph. Sometimes two mass spectrometers are paired, so that a mild ionization method can be followed by a more vigorous ionization of the individual fragments.

See also Spectroscopy.

mass spectrometry Technique that allows the measurement of atomic and molecular masses. Material is vaporized in a vacuum, ionized, and then passed first through a strongly accelerating electric potential and then through a powerful magnetic field. This serves to separate the ions in order of their charge: mass ratio; the ions are detected, commonly by means of an electrometer which measures the force between charges and hence the electric potential. Mass spectrometry is used in the radiometric dating of rocks, and in isotope geochemistry.