Figure 8–17 Western blotting. All the proteins from dividing tobacco cells in culture were first separated by two-dimensional polyacrylamide-gel electrophoresis. In (A), the positions of the proteins are revealed by a sensitive protein stain. In (B), the separated proteins on an identical gel were then transferred to a sheet of nitrocellulose and exposed to an antibody that recognizes only those proteins that are phosphorylated on threonine residues during mitosis. The positions of the few proteins that are recognized by this antibody are revealed by an enzyme-linked second antibody. (From
J.A. Traas et al., Plant J. 2:723–732, 1992. With permission from Blackwell Publishing.)
Figure 8–18 The mass spectrometer. (A) Mass spectrometers used in biology contain an ion source that generates gaseous peptides or other molecules under conditions that render most molecules positively charged. The two major types of ion source are MALDI and electrospray, as described in the text. Ions are accelerated into a mass analyzer, which separates the ions on the basis of their mass and charge by one of three major methods: 1. Time-of-flight (TOF) analyzers determine the massto-charge ratio of each ion in the mixture from the rate at which it travels from the ion source to the detector. 2. Quadropole mass filters contain a long chamber lined by four electrodes that produce oscillating electric fields that govern the trajectory of ions; by varying the properties of the electric field over a wide range, a spectrum of ions with specific mass-to-charge ratios is allowed to pass through the chamber to the detector, while other ions are discarded. 3. Ion traps contain doughnut-shaped electrodes producing a three-dimensional electric field that traps all ions in a circular chamber; the properties of the electric field can be varied over a wide range to eject a spectrum of specific ions to a detector. (B) Tandem mass spectrometry typically involves two mass analyzers separated by a collision chamber containing an inert, high-energy gas. The electric field of the first mass analyzer is adjusted to select a specific peptide ion, called a precursor ion, which is then directed to the collision chamber. Collision of the peptide with gas molecules causes random peptide fragmentation, primarily at peptide bonds, resulting in a highly complex mixture of fragments containing one or more amino acids from throughout the original peptide. The second mass analyzer is then used to measure the masses of the fragments (called product or daughter ions). With computer assistance, the pattern of fragments can be used to deduce the amino acid sequence of the original peptide.
sequences present in the unknown sample with known cataloged genes. This task is now performed almost exclusively by using mass spectrometry in conjunction with computer searches of databases.
Charged particles have very precise dynamics when subjected to electrical and magnetic fields in a vacuum. Mass spectrometry exploits this principle to separate ions according to their mass-to-charge (m/z) ratio. It is an enormously sensitive technique. It requires very little material and is capable of determining the precise mass of intact proteins and of peptides derived from them by enzymatic or chemical cleavage. Masses can be obtained with great accuracy, often with an error of less than one part in a million.
Mass spectrometry is performed using complex instruments with three major components (Figure 8–18A). The first is the ion source, which transforms tiny amounts of a peptide sample into a gas containing individual charged peptide molecules. These ions are accelerated by an electric field into the second component, the mass analyzer, where electric or magnetic fields are used to separate the ions on the basis of their mass-to-charge ratios. Finally, the separated ions collide with a detector, which generates a mass spectrum containing a series of peaks representing the masses of the molecules in the sample.