The rapid development in proteomics over the last 10 years has led to a series of new technologies and combinations of them designed to unravel as much as possible of the proteins of an organism or otherwise specified biological material. Despite being a little tricky at certain steps, 2-DE has a very high resolution power with more than 10,000 spots per gel and is able to separate one protein into its different protein species caused by posttranslational modifications, alternative splicing or genetic variability. This high-resolution separation is combined with a highly sensitive identification method using peptide mass fingerprinting combined with sequence information by MS/MS, which results in high sequence coverage: the key to elucidate protein species structures. The off-line measurement by MALDI-TOFTOF-MS allows the repeated measurement of each sample and therefore provides more complete structure information for each protein species. The presented protocols represent the basic technology consisting of 2-DE, two staining methods, tryptic digestion and MALDI-TOFTOF-MS.
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