Proteomics

The most basic method used in proteomics is a twodimensional (2D) electrophoresis. Cellular or tissue extracts are separated on a polyacrylamide gel in two dimensions, according to their charge and size, producing a pattern of spots. Although up to 11,000 spots can be separated on one gel, a typical number is approximately 2000. Following the separation, patterns obtained from test and control samples are overlaid and analyzed to determine any changes in protein expression, their levels or modifications. Proteins that are present in one, but not the other sample are isolated from the gel. In order to identify them, proteins are digested with an enzyme (usually trypsin) and the obtained small fragments (peptides) are analyzed by mass spectrometry to produce peptide fingerprints or protein tags that can be used for identification of the unknown spot. A second method used in analysis is tandem spectrometry. When each of the analyzed peptides are further digested and re-analyzed, this approach produces some sequence information in addition to mass. It is important to realize that mass spectrometry or even microsequencing are not used to fully sequence the samples, but to create sufficient information that will identify the unknown protein by searching the databases.

An important area of proteomic studies is to identify the interactions between proteins to determine the networks created by proteins in cells. A method used for such studies is a yeast two-hybrid system, which is best compared to fishing. Scientists use a bait molecule (chimeric protein), produced from the DNA sequence of a protein of interest fused to a sequence of a DNA-binding part of a known transcriptional activator, to identify what protein (prey molecule) interacts with their protein of interest. The process of identification of interacting complexes involves observing a color change resulting from an activation of a reporter gene. This gene is activated by a formed complex due to the fact that a prey molecule contains a coding sequence of a protein that might interact with the bait fused to an activation domain of the same transcriptional activator used for creating the bait. As of 2002, a number of commercial companies (for example, Hybrigenics) have developed a large scale automated yeast two hybrid screen.

An alternative method for studying protein interactions is creating tagged proteins, introducing them into cells, and subsequently using the tag to isolate the protein complexes formed in cells. The complexes are then separated on a gel according to their size and individual proteins are isolated, and identified by mass spectrometry.

Computational tools in proteomics are very important as the data generated requires image analysis, peptide and protein tag analysis, extensive database searching, and further investigation involving for example protein modification analysis.