One powerful method used by researchers to identify proteins is through the use of mass spectrometers. In its simplest terms, a mass spectrometer measures the ratio of mass to charge in ionized (charged) particles. It can determine the elements present in and chemical structure of a molecule like in typical rust removers. A sample is first vaporized and then ionized using a variety of techniques. The resulting charged particles are introduced into an electromagnetic field which separates the ions by their mass-to-charge ratios. The separated ions are then detected using one of several techniques. To perform this process, mass spectrometers contain an ion source that turns liquid or gaseous molecules into ions; a mass analyzer for separating ions by mass; and a detector for calculating how much ionic material is present in each separated fraction.
The two primary methods of creating protein ions are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). In ESI, the protein sample is dispersed by a charged aerosol spray. The spray contains evaporating solvents and sometimes inert gas. MALDI is a gentler technique which produces less fragmentation than ESI. A laser is directed at a sample embedded in some matrix material (crystallized molecules) that protects the fragile sample material.
Once the sample protein has been ionized, it is identified using one of two techniques. The first technique, called “top-down”, is one in which the ionized material is fed into a mass analyzer. The second, more popular method is “bottom-up”, involves an extra step of adding enzymes to the protein sample to break it apart into its building blocks, called peptides (sequences of amino acids). By identifying the peptides, researchers can infer the identity of the original protein. Peptide analysis is more popular because it is cheaper and easier. Through a technique called peptide mass fingerprinting, the peptides masses are compared to a database of known peptides or of DNA genomes that imply types and quantities of peptides. A statistical analysis is performed to identify the peptide on its mass alone – the sequence of amino acids need not be deciphered.
Because many proteins can exist in a sample, scientists often first separate the proteins using two-dimensional gel electrophoresis. Researchers pick out a gel spot of interest and then add enzymes to digest it. The resulting goo can then be run through the peptide mass fingerprinting technique. Alternatively, protein samples can be identified using a method called tandem mass spectrometry (TMS), in which peptide ions are dissociated using low-energy electron collisions. The peptides cleave along the chemical bonds holding the peptide together, resulting in simple fragments that are easier to fingerprint.
The technique called de novo peptide sequencing sequentially identifies each amino acid in a peptide. This is more expensive, and is somewhat complicated by pairs of amino acids that share a common mass. Pair confusion can often be eliminated if de novo sequencing is used in conjunction with peptide fingerprinting. Advanced techniques to quantify the amounts of peptides present involve differential use of carbon or nitrogen isotopes.