Establishment of a High Performance UPLC-QSTAR Elite Proteomics Platform at Unive

描述（由申请人提供）：此共享仪器应用寻求建立高性能现代蛋白质组学平台的资金，以加强佛罗里达大学（UF）的生物医学研究。该平台由Applied Biosystems新一代QSTAR Elite四极杆飞行时间（QTOF）质谱仪和相关的纳流二维超高效液相色谱（UPLC）（由沃茨公司制造）组成。ProteinPilot 2.0软件包进行数据分析。我们建议操作这种集成的nanoflow UPLC-QTOF MS/MS系统，用于分离和分析复杂的肽（和蛋白质）混合物，作为20多个NIH资助的赠款的一大组的一部分，从中选择了8个代表性的组，以证明对最先进的蛋白质组学技术的迫切需求。这个新系统的最关键的特点是：在前端分离和质谱的数量，可以获得显着的改善，整体灵敏度，分辨率和再现性，动态范围，低丰度肽的表征，智能碰撞能量，离子传输/积累，和定量软件功能。该蛋白质组学平台的独特功能将针对大量蛋白质和蛋白质组学应用，其中LC-MS用于蛋白质/肽鉴定，复杂混合物的准确定量，以及生物学上重要的翻译后修饰的测定，如蛋白质磷酸化。由于基于二维凝胶的蛋白质组学的局限性，近年来已经看到对使用或不使用同位素标记实现多维LC-MS的鸟枪法蛋白质组学的需求日益增加。这种技术已被证明在扩大蛋白质组覆盖范围和鉴定低丰度蛋白质方面是有效的。然而，在每个实验的基础上，它需要大量的仪器时间。由于需求不断增长，我们蛋白质组学设施中现有的LC-MS仪器的使用受到限制，而新技术的性能优于跨学科生物技术研究中心（ICBR）蛋白质组学部门的现有仪器，该中心是佛罗里达大学的主要研究服务设施校园。因此，我们建议建立这个新的技术平台，为iTRAQ实验和鸟枪蛋白质组学提供更快的速度和性能，同时补充设施中的现有技术，并提高NIH项目的样品通量，实验设计和生产力，这些项目正在研究与人类健康密切相关的关键问题，包括神经退行性疾病，脑损伤，癌症，生物标志物发现环境毒理学，和传染病。公共卫生相关性：许多人类疾病和紊乱直接或间接由蛋白质功能障碍引起。蛋白质生物标志物，其中许多以低量和分子的复杂混合物存在，指示疾病状态和/或负责疾病发展。获得高性能蛋白质组学平台将极大地促进美国国立卫生研究院资助的佛罗里达大学和vinicity机构的研究人员在疾病的早期阶段检测，识别和量化蛋白质生物标志物，为疾病预防，治疗和药物开发提供有效方法，从而大大改善公共健康。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.