The objective of this grant is to purchase an integrated instrumentation system that includes high-resolution and high-throughput, two-dimensional electrophoresis and dedicated mass spectrometry to analyze patterns of protein expression (proteomics) relevant to human disease. The requested instrumentation will provide state-of-the-art proteomics analytical capabilities for a large NIH-funded biomedical research community at the University of Alabama at Birmingham. Analysis of single protein changes and modifications has been a major utilization of the mass spectrometry facility, principally through use of the matrix-assisted laser-desorption ionization-time-of-flight mass spectrometer (MALDI-TOF MS), and the Q-TOF. In this postgenome era, once a polypeptide gel """"""""spot"""""""" is excised, trypsin-digested and analyzed by MALDI-TOF, the availability of genome sequence data enables the rapid identification of unknown polypeptides through various software. The requested instrumentation will address the major factors that limit proteomic analysis: (1) enhance the detection of a relevant polypeptide(s) in a 2-D gel """"""""display,"""""""" (2) allow greater resolution of a display for a given sample, and (3) enable high-throughput processing of gel """"""""spots"""""""" such that this step is not rate limiting. Accompanying software ensure seamless integration among various instruments, and allow sample tracking from beginning step, isoelectric focusing, to the last step, MALDI-TOF identification of protein based on automated peptide mass fingerprinting. In the last year, the principal investigator directed an initial developmental phase of 2-D electrophoresis in conjunction with the mass spectrometry facility, utilizing mini gels and Coomassie-or silver stained mini gels for the second dimension. The conclusion is that it is essential to go to larger-format gels, with fluorescence detection, robotic excision and in-gel digestions of gel """"""""spots,"""""""" as well as spotting onto MALDI-TOF plates, ending with automated MALDI-TOF mass spectrometry The requested integrated system (BioRad-Micromass) will provide the instrumentation to take our proteomics analytical capabilities to the level that will sustain the high quality of biomedical research that is a hallmark of UAB.
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