Progress, and Applications
of the Human Genome Project
Sponsored by the U.S. Department of Energy Human Genome Program
Human Genome News Archive Edition
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In the News
Genetics in Medicine
2-DGE: A Technique for Visualizing Protein Expression and Modification
One current proteomic tool for visualizing and quantitating all proteins expressed in a biological system at a given time is two-dimensional gel electrophoresis (2-DGE). As originally described by Patrick O'Farrell for analyzing Escherichia coli proteins in 1975, 2-DGE combines the electrophoretic separation of denatured proteins by isoelectric point charge differences in the first dimension with separation based on molecular size differences in the second dimension. The proteins, which can be detected using protein-specific stains, appear as constellations of spots in the 2-D space of the gel. Over the 20-year history of 2-DGE, numerous algorithms have been developed for comparing 2-DGE patterns and quantitatively analyzing protein abundance.
The recent addition of mass spectrometry to methods available for identifying proteins detected by 2-DGE has provided the needed capability for rapid identification. Proteins can be digested in the 2-DGE gel using a specific protease (e.g., trypsin or amino acid-specific endopeptidases), the resulting peptides eluted, and the masses of proteins determined using mass spectrometry. [Matrix-assisted laser desorption ionization (MALDI-MS) and electrospray currently are the preferred methods.] The peptide masses are then used to search protein and DNA sequence databases for the identity of predicted amino acid sequences to produce the same peptide masses when cleaved with the same protease. When a complete genome sequence is available and the peptide mass search is limited to just that sequence database, the protein identification process is highly reliable and efficient.
The work of John Yates's group at the University of Washington, in which 260 Haemophilus influenzae proteins separated by 2-DGE were identified in about a month with MALDI-MS, demonstrates this approach's potential for identifying the hundreds of proteins revealed in the 2-DGE patterns of cell lysates. The protein-expression information can then be compared to the cell's proteome and other proteomes to provide a better understanding of cell function.
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Last modified: Wednesday, October 29, 2003
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