In a simple, single quadrupole mass spectrometer, a sample is placed under vacuum and allowed to vaporize in the presence of a proton donor to impart a positive charge. An electrical field then propels the cations toward a curved flight tube where they encounter a magnetic field, which deflects them at a right angle to their original direction of flight (Figure 1). The current powering the electromagnet is gradually increased until the path of each ion is bent sufficiently to strike a detector mounted at the end of the flight tube. For ions of identical net charge, the force required to bend their path to the same extent is proportionate to their mass.

Fig1. Basic components of a simple mass spectrometer. A mixture of molecules, represented by a red circle, green triangle, and blue diamond, is vaporized in an ionized state in the sample chamber. These molecules are then accelerated down the flight tube by an electrical potential applied to the accelerator grid (yellow). An adjustable field strength electromagnet applies a magnetic field that deflects the flight of the individual ions until they strike the detector. The greater the mass of the ion, the higher the magnetic field required to focus it onto the detector.

Time-of-flight (TOF) mass spectrometers employ a linear flight tube. Following vaporization of the sample in the presence of a proton donor, an electric field is briefly applied to accelerate the ions toward a detector at the end of the flight tube. For molecules of identical charge, the velocity to which they are accelerated, and hence the time required to reach the detector, is inversely proportional to their mass.

Quadrupole mass spectrometers are generally used to determine the masses of molecules of 4000 Da or less, whereas TOF mass spectrometers are used to determine the large masses of complete proteins. Various combinations of multiple quadrupoles, or reflection of ions back down the linear flight tube of a TOF mass spectrometer, are used to create more sophisticated instruments.

Peptides Can Be Volatilized for Analysis by Electrospray Ionization or Matrix-Assisted Laser Desorption

 For many years, the analysis of peptides and proteins by MS initially was hindered by difficulties in volatilizing these large organic molecules. While small organic molecules could be readily vaporized by heating in a vacuum (Figure 2), proteins, oligonucleotides, etc. decomposed on heating. Only when reliable techniques were devised for dispersing peptides, proteins, and other large biomolecules into the vapor phase was it possible to apply MS for their structural analysis and sequence determination. Three commonly used methods for dispersion into the vapor phase are electrospray ionization, matrix-assisted laser desorption and ionization (MALDI), and fast atom bombardment (FAB). In electrospray ionization, the molecules to be analyzed are dissolved in a volatile solvent and introduced into the sample chamber in a minute stream through a charged capillary probe (see Figure2). As the droplet of liquid emerges into the sample chamber, the charged probe ionizes the sample while the solvent rapidly disperses, leaving the macromolecule suspended in the gaseous phase. Electrospray ionization is frequently used to analyze peptides and proteins as they elute from an HPLC or other chromatography column, already dissolved in a volatile solvent. In MALDI, the sample is mixed with a liquid matrix containing a light-absorbing dye and a source of protons. In the sample chamber, the mixture is excited using a laser, causing the surrounding matrix to disperse into the vapor phase so rapidly as to avoid heating embedded peptides or proteins (see Figure 2). In FAB, large macromolecules dispersed in glycerol or another protonic matrix are bombarded by a stream of neutral atoms, for example, xenon, that have been accelerated to a high velocity. “Soft” ionization by FAB is frequently applied to volatilize large macromolecules intact.

Fig2. Three common methods for vaporizing molecules in the sample chamber of a mass spectrometer.

Peptides inside the mass spectrometer can be broken down into smaller units by collisions with neutral helium or argon atoms (collision-induced dissociation) and the masses of the individual fragments determined. Fortunately, since peptide bonds are more vulnerable to rupture than carbon-carbon bonds, the most abundant fragments will differ from one another by increments of one or two amino acids. Since— with the exceptions of (1) leucine and isoleucine and (2) glutamine and lysine—the molecular mass of each amino acid is unique, the sequence of the peptide can be reconstructed from the masses of its fragments.

Tandem Mass Spectrometry

Complex peptide mixtures can be analyzed, without prior purification, by tandem MS, which employs the equivalent of two mass spectrometers linked in series. For this reason, analysis by tandem instruments is often referred to as MS–MS, or MS2. The first mass spectrometer separates individual pep tides based on their differences in mass. By adjusting the field strength of the first magnet, a single peptide can be directed into the second mass spectrometer, where fragments are generated and their masses are determined. Alternatively, they can be held in an electromagnetic ion trap located between the two quadrupoles and selectively delivered to the second quadrupole instead of being lost when the first quadrupole is set to select ions of a different mass.

Tandem MS can be used to screen blood samples from newborns for the presence and concentrations of amino acids, fatty acids, and other metabolites. Abnormalities in metabolite levels can serve as diagnostic indicators for a variety of genetic dis orders, such as phenylketonuria, ethylmalonic encephalopathy, and glutaric acidemia type 1. In recent years a new generation of tandem mass spectrometers has appeared, called Q-TOF-MS, that couple quadrupole (Q) and time-of-flight (TOF) technologies together in order to harness the best aspects of each.

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