This application is for funds to purchase the Micromass QTOF Ultima API Hybrid Quadrupole/Orthogonal Time of Flight Mass Spectrometer. At the present time the University of Florida does not have this type of instrument on campus and is not able to support research requiring this instrumentation. The QTOF is state-of-the art technology for identifying proteins and post-translational modifications in proteomics projects. The main features we require include an electrospray ionization source on a quadrupole/orthogonal time-of-flight mass spectrometer, the ability to combine capillary- and nano-HPLC on the front end with the electrospray function, tandem mass spectrometry (MS/MS), high mass range, high resolution, high mass accuracy (5-10 ppm), fast scan rate (10 scans/sec), determination of exact mass of precursor and neutral loss molecules in MS/MS, and good software that is intuitive and easy to use. The QTOF excels in each of these areas and thus is the instrument we have chosen. We will be able to solve difficult protein identification problems and obtain de novo sequence information from protein samples that are obtainable only in fmol amounts. We will also be able to pinpoint positions of post-translational modification. The projects proposed require mass spectrometry to answer important questions about the identity and the regulation of proteins in important steps in the differentiation of tissues and in important biochemical pathways. Two of the projects (Prokai and Dunn) aim to use combinatorial approaches for finding the best substrates for proteases that are involved in apartic protease diseases or as substrate for brain proteases involved in making neuropeptides. One project (West) has already applied QTOF mass spectrometry to begin to characterize proteins that are involved in unique cytoplasmic glycosylation reactions. He seeks to continue these studies with the new instrument. One project (Sugrue) will seek to identify proteins that are involved in complexes with the tumor suppressor gene pinin. One project (May) is involved in understanding the role that proteins play in apoptosis, still another (Moyer) is looking to define the structure of poxvirus encoded serpins. (Hargrave) is looking to better understand the proteins that bind to rhodopsin and the role of adhesion related motecutes in corneal epithelial integrity. One project (Petersen) is involved in characterizing protein signals involved in defining stem cells and their differentiation into tissues. One project (Hayes) will characterize proteins that are altered after traumatic brain injury. All of these projects will result in better understanding of fundamental processes that lead to the potential treatment of diseases.
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