: The Core will serve all projects in this Program. The use of 2D gels and mass spectrometry (MS) represents the most efficient way to perform proteomic analyses. It is now possible to rapidly characterize 1,000-3,000 of the cellular proteins and to compare protein expression after specific manipulations of the cells. The Core will provide methodology for proteomics analysis based on 2-D gel electrophoresis coupled with in-gel digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF) or liquid chromatography/tandem mass spectrometry (LC/MS/MS). Protein spots will be imaged and stored so that they can be compared with a single standard image. All spots will be excised from the gel but identification of differentially expressed spots will be the major goal of analyses that are performed. The other spots will be stored at -70 degree C in case other targets are discovered during subsequent research. The proteins present in gel cores will be subjected to in-gel digestion with subsequent extraction of the peptide fragments for analysis by MS. In-gel digestion will normally be conducted with trypsin using 96-wellplates. Samples will be de-salted prior to MS analyses using commercially available micro columns. The samples will then be spotted on MALDI-TOF targets or injected directly on an LC/MS system. In a typical proteome analysis, MALDI-TOF/MS will be conducted on peptide digests of the excised gel spots to rapidly obtain partial sequences. Mass measurements will be conducted with accuracy in the 5-10 ppm range by using internal calibration of protease fragments present in the incubation. This method will be particularly useful for proteins that appear in databases as it is possible to identify a protein with as few as 3-5 peptide fragments. For unknown peptides, this approach has only limited applicability. It will be necessary to use LC/MS/MS for the full structural characterization of unknown proteins. MS/MS analyses will also be conducted when necessary using partially purified peptide fragments in combination with nanospray infusions. Sequencing of unknown peptides will be performed using computer programs such as Sequest, MASCOT, peptidesearch, PROWL, and protein. The Sequest system works particularly well with marginal MS/MS data but does not allow searching on the internet. MASCOT will therefore serve as an ideal adjunct for web searching. This Core is an absolutely vital component of the PPG.
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