f IC U R E 20-24 Analysis of the proteome by 20 electrophoresis and mass spectrometry, (a)

Example of proteins from a ceil extract separated by 2D gel electrophoresis. Note that in this example, only proteins with a small range of isoelectric points (between 5 and 5.5) are analyzed (here separated left to right). IIF stands for isoelectric focusing. The vertical direction separates proteins by their SDS-denatured molecular weight, fcach dark spot usually represents a single pro tern (although or* occasion individual protein spots overlap), (b) This pane! shows a close up of a smalt segment of the gel in (a) The large protein spot in the middle ts selected for further analysis. The gel slice is treated with trypsin, which deanes the polypeptide chain after each of its positively-charged amino aods {K or R). These peptides are then eluted from the gel, and analyzed by mass spectrometry, (c) An example spectrum in which individual peptides are separated from one another by their signature mass to charge ratio. (Source: Parts a and b are reproduced, with permission, from Simpson R.J. 2003. Prolans and proteomics: A laboratory manual, p 555. fig 6.47, parts a and b Cold Spring Harbor Laboratory Press, Cold Spring Harbor NV)

acting partner proteins. Whereas microarray analysis (see Chapter 18) makes it possible to visualize gene transcription on a genome-wide basis, the tools of proteomics provide r snapshot of the cell's full repertoire of proteins.

Proteomics is based on three principal methods: two-dimensional gel electrophoresis for protein separation, mass spectrometry for the precise determination of the molecular weight and identity of a protein (or peptides generated from the protein), and bioinformatics for assigning proteins and peptides to the predicted products of protein-coding sequences in the genome. A single cell often produces thousands of different proteins, far too many to separate and identify by SDS gel electrophoresis alone. As its name implies, two-dimensional gel electrophoresis separates proteins in two dimensions and does so in successive stops.

In the first step, the proteins are fractionated according to their isoelectric point by isoelectric focusing. During isoelectric focusing, a gradient of pH is generated in a gel. The isoelectric point is the pH at which a protein exhibits no net charge and hence becomes stationary (focuses) in the pH gradient. In the second step, the proteins an: separated according to size by SDS gel electrophoresis as described above. Because proteins are separated on the basis of two properties (isoelectric point and molecular weight], thousands of different proteins can be resolved from each other in a single experiment. After fractionation by two-dimensional gel electrophoresis, each protein is separately subjected to mass spectrometry in order to determine its exact molecular weight. As discussed above, it is generally more effective to first treat the protein with a protease and then determine the molecular weight of the resulting proteolytic fragments, rather than the intact protein itself. MS/MS analysis also allows the precise sequence of the polypeptide fragments of each protein to be identified.

Finally, given a complete genome sequence for the organism under study and these peptide sequences from the proteins of interest, the tools of bioinformatics make it possible to assign each protein (that is, its proteolytic fragments) to a particular protein-coding sequence (gene) in the genome.

Was this article helpful?

0 0

Post a comment