There are many different types of mass spectrometer, varying mainly in the nature of their ion sources and mass analyzers. One of the most common ion sources depends on a technique called matrix-assisted laser desorption ionization (MALDI). In this approach, the proteins in the sample are first cleaved into short peptides by a protease such as trypsin. These peptides are mixed with an organic acid and then dried onto a metal or ceramic slide. A brief laser burst is directed toward the sample, producing a gaseous puff of ionized peptides, each carrying one or more positive charges. In many cases, the MALDI ion source is coupled to a mass analyzer called a time-of-flight (TOF) analyzer, which is a long chamber through which the ionized peptides are accelerated by an electric field toward a detector. Their mass and charge determine the time it takes them to reach the detector: large peptides move more slowly, and more highly charged molecules move more quickly. By analyzing those ionized peptides that bear a single charge, the precise masses of peptides present in the original sample can be determined. This information is then used to search genomic databases, in which the masses of all proteins and of all their predicted peptide fragments have been tabulated from the genomic sequences of the organism. An unambiguous match to a particular open reading frame can often be made by knowing the mass of only a few peptides derived from a given protein.
By employing two mass analyzers in tandem (an arrangement known as MS/ MS; Figure 8–18B), it is possible to directly determine the amino acid sequences of individual peptides in a complex mixture. The MALDI-TOF instrument described above is not ideal for this method. Instead, MS/MS typically involves an electro-spray ion source, which produces a continuous thin stream of peptides that are ionized and accelerated into the first mass analyzer. The mass analyzer is typically either a quadropole or ion trap, which employs large electrodes to produce oscillating electric fields inside the chamber containing the ions. These instruments act as mass filters: the electric field is adjusted over a broad range to select a single peptide ion and discard all the others in the peptide mixture. In tandem mass spectrometry, this single ion is then exposed to an inert, high-energy gas, which collides with the peptide, resulting in fragmentation, primarily at peptide bonds. The second mass analyzer then determines the masses of the peptide fragments, which can be used by computational methods to determine the amino acid sequence of the original peptide and thereby identify the protein from which it came.
Tandem mass spectrometry is also useful for detecting and precisely mapping post-translational modifications of proteins, such as phosphorylations or acetylations. Because these modifications impart a characteristic mass increase to an amino acid, they are easily detected during the analysis of peptide fragments in the second mass analyzer, and the precise site of the modification can often be deduced from the spectrum of peptide fragments.
A powerful, “two-dimensional” mass spectrometry technique can be used to determine all of the proteins present in an organelle or another complex mixture of proteins. First, the mixture of proteins present is digested with trypsin to produce short peptides. Next, these peptides are separated by automated high-performance liquid chromatography (LC). Every peptide fraction from the chromatographic column is injected directly into an electrospray ion source on a tandem mass spectrometer (MS/MS), providing the amino acid sequence and post-translational modifications for every peptide in the mixture. This method, often called LC-MS/MS, is used to identify hundreds or thousands of proteins in complex protein mixtures from specific organelles or from whole cells. It can also be used to map all of the phosphorylation sites in the cell, or all of the proteins targeted by other post-translational modifications such as acetylation or ubiquitylation.
Sets of Interacting Proteins Can Be Identified by Biochemical Methods
Because most proteins in the cell function as part of complexes with other proteins, an important way to begin to characterize the biological role of an unknown protein is to identify all of the other proteins to which it specifically binds.