This shared instrumentation application seeks funding for the establishment of a high performance modern proteomics platform to enhance biomedical research at the University of Florida (UF). The platform consists of an Applied Biosystems new generation QSTAR Elite quadrupole time-of-flight (QTOF) mass spectrometer and associated nanoflow two-dimensional ultra performance liquid chromatography (UPLC) (manufactured by Waters Inc.) and ProteinPilot 2.0 software package for data analysis. We propose to operate this integrated nanoflow UPLC-QTOF MS/MS system for separation and analysis of complex peptide (and protein) mixtures as part of a large group of over 20 NIH funded grants, from which eight representative groups were selected to demonstrate the pressing needs for the state-of-the-art proteomics technology. The most critical features of this new system are: significant improvement in front-end separation and the number of mass spectra that can be acquired, overall sensitivity, resolution and reproducibility, dynamic range, characterization of low abundance peptides, smart collision energetics, ion transmission/accumulation, and quantitation software features. The unique capabilities of this proteomics platform will be directed at a large array of protein and proteomic applications where LC-MS is used for protein/peptide identification, accurate quantitation of complex mixtures, as well as the determination of biologically significant posttranslational modifications, such as protein phosphorylation. Because of the limitations of 2D gel based proteomics, recent years have seen increasing demands for shotgun proteomics implementing multiple dimension LC-MS with or without isotope labeling. Such technology has been proven effective in expanding the proteome coverage and identifying low abundance proteins. However, it requires significant instrument time on a per experiment basis. Access to the existing LC-MS instrument in our Proteomics facility is limited due to the growing demands, and newer technologies outperform existing instruments in the Proteomics Division at the Interdisciplinary Center for Biotechnology Research (ICBR), the major research service facility on the UF campus. Therefore, we propose to establish this new technical platform to provide greater speed and performance for iTRAQ experiments and shotgun proteomics while complementing existing technologies in the facility and improving the sample throughput, experimental design, and productivity of the NIH projects that are examining key issues closely related to human health, including neurodegenerative disease, brain injury, cancer, biomarker discovery environmental toxicology, and infectious diseases.
Many human diseases and disorders are directly or indirectly caused by protein dysfunction. Protein biomarkers, many of which are present in low amounts and in a complex mixture of molecules, are indicative of disease states and/or are responsible for disease development. Acquisition of a high performance proteomics platform will greatly facilitate the NIH funded researchers at the University of Florida and the vinicity institutions to detect, identify and quantify the protein biomarkers at an early stage of disease, inform effective approaches to disease prevention, treatment and drug development, and thus greatly improve public health.
