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.